Anti-human VISTA antibodies and use thereof

ABSTRACT

The invention provides antagonistic and agonistic anti-human VISTA antibodies and antibody fragments. These antagonist antibodies and antibody fragments may be used to inhibit or block VISTA&#39;s suppressive effects on T cell immunity and thereby promote T cell immunity. These agonist antibodies and antibody fragments may be used to potentiate or enhance or mimic VISTA&#39;s suppressive effects on T cell immunity and thereby suppress T cell immunity. These antagonist antibodies and antibody fragments are especially useful in the treatment of cancer and infectious conditions. These agonist antibodies and antibody fragments are especially useful in the treatment of autoimmunity, allergy, inflammatory conditions, GVHD, sepsis and transplant recipients. Screening assays for identifying these agonists are also provided.

RELATED APPLICATIONS

This application is a U.S. National Phase Application submitted under 35U.S.C. 371 based on International Application No. PCT/US2017/027765filed Apr. 14, 2017 (published as WO/2017/181109 on Oct. 19, 2017). Thisapplication is also a Continuation-in-part of International ApplicationNo. PCT/US17/027800 filed Apr. 14, 2017 (published as WO/2017/181139 onOct. 19, 2017). Both International Applications PCT/US2017/027765 andPCT/US17/027800 claim priority to the following U.S. ProvisionalApplications: U.S. Prov. Appl. No. 62/323,193 filed Apr. 15, 2016; U.S.Prov. Appl. No. 62/343,355 filed May 31, 2016; U.S. Prov. Appl. No.62/363,929 filed Jul. 19, 2016; U.S. Prov. Appl. No. 62/363,931 filedJul. 19, 2016; U.S. Prov. Appl. No. 62/363,917 filed Jul. 19, 2016; U.S.Prov. Appl. No. 62/364,073 filed Jul. 19, 2016; U.S. Prov. Appl. No.62/363,925 filed Jul. 19, 2016; U.S. Prov. Appl. No. 62/365,085 filedJul. 21, 2016; U.S. Prov. Appl. No. 62/365,102 filed Jul. 21, 2016; U.S.Prov. Appl. No. 62/365,081 filed Jul. 21, 2016; U.S. Prov. Appl. No.62/365,166 filed Jul. 21, 2016; U.S. Prov. Appl. No. 62/365,087 filedJul. 21, 2016; U.S. Prov. Appl. No. 62/372,362 filed Aug. 9, 2016; U.S.Prov. Appl. No. 62/385,627 filed Sep. 9, 2016; U.S. Prov. Appl. No.62/385,805 filed Sep. 9, 2016; U.S. Prov. Appl. No. 62/385,871 filedSep. 9, 2016; U.S. Prov. Appl. No. 62/385,888 filed Sep. 9, 2016; U.S.Prov. Appl. No. 62/385,893 filed Sep. 9, 2016; U.S. Prov. Appl. No.62/385,785 filed Sep. 9, 2016; U.S. Prov. Appl. No. 62/406,632 filedOct. 11, 2016; U.S. Prov. Appl. No. 62/425,184 filed Nov. 22, 2016, eachand all of which are hereby incorporated by reference in theirentireties.

SEQUENCE LISTING DISCLOSURE

This application includes as part of its disclosure a biologicalsequence listing which is being concurrently submitted through EFS-Web.Said biological sequence listing is contained in a file named“43260o2301.txt” which was created on Oct. 15, 2018, and has a size of536,228 bytes, and is hereby incorporated by reference in its entirety.

FIELD

The invention relates to the identification of novel anti-human VISTAantibodies and antibody fragments, i.e., anti-human VISTA (V-regionImmunoglobulin-containing Suppressor of T cell Activation(1)), (“VISTA”)antibodies and antibody fragments. More specifically, the presentapplication provides novel human VISTA agonists, i.e., anti-human VISTAantibodies and antibody fragments which agonize or promote thesuppressive effects of human VISTA on immunity, particularly T cellimmunity. Also, the invention relates to the use of such agonists toenhance or mimic the suppressive effects of VISTA on immunity such asits suppressive effects on CD4⁺ or CD8⁺ T cell proliferation, CD4⁺ orCD8⁺ T cell activation and its suppressive effect on the production ofimmune cytokines, particularly proinflammatory cytokines. Also theinvention relates to the specific use of these agonistic antibodies andantibody fragments as prophylactics or therapeutics, especially intreating conditions wherein the prevention or inhibition of T cellimmunity and the expression of proinflammatory cytokines istherapeutically beneficial such as autoimmunity, inflammation, allergicdisorders, sepsis, GVHD or in alleviating the inflammatory side effectsof some conditions such as cancer.

The present application also provides novel antagonists, i.e.,anti-human VISTA antibodies and antibody fragments which antagonize orinhibit the suppressive effects of human VISTA on immunity, particularlyVISTA's effects on T cell immunity. Also, the invention relates to theuse of such novel antagonists to block or inhibit the suppressiveeffects of VISA on immunity, i.e., its suppressive effects on CD4⁺ orCD8⁺ T cell proliferation, CD4⁺ or CD8⁺ T cell activation and theproduction of immune cytokines. Also the invention also relates to thespecific use of these antagonistic antibodies and antibody fragments asprophylactics or therapeutics, especially in treating conditions whereinpromoting T cell immunity is therapeutically beneficial such as in thetreatment of cancer and infectious diseases.

BACKGROUND

Immune negative checkpoint regulator (NCR) pathways have proven to beextraordinary clinical targets in the treatment of human immune-relateddiseases. Blockade of two NCRs, CTLA-4 and PD-1, using monoclonalantibodies (mAbs) to enhance tumor immunity is revolutionizing thetreatment of cancer and has established these pathways as clinicallyvalidated targets in human disease. Also soluble versions of NCR ligandsthat trigger NCR pathways have entered the clinic as immunosuppressivedrugs to treat autoimmunity (i.e., AMP-110/B7-H4-Ig for Rheumatoidarthritis).

VISTA (see Ref 1), is an NCR ligand, whose closest phylogenetic relativeis PD-LI. VISTA bears homology to PD-LI but displays a unique expressionpattern that is restricted to the hematopoietic compartment.Specifically, VISTA is constitutively and highly expressed onCDIIb^(hlgh) myeloid cells, and expressed at lower levels on CD4⁺ andCD8⁺ T cells. Like PD-LI, VISTA is a ligand that profoundly suppressesimmunity (Ref 1), and like PD-LI, blocking VISTA allows for thedevelopment of therapeutic immunity to cancer in pre-clinical oncologymodels (see Ref 2). Whereas blocking VISTA enhances immunity, especiallyCD8⁺ and CD4⁺ mediated T cell immunity, treatment with a soluble Igfusion protein of the extracellular domain of VISTA (VISTA-Ig)suppresses immunity and has been shown to arrest the progression ofmultiple murine models of autoimmune disease.

Clear scientific evidence has shown that VISTA is a ligand that inducesprofound T cell suppression. Numerous antagonistic anti-human VISTAantibodies have been reported by different groups including DartmouthCollege and Jannsen. These antibodies are useful in the treatment ofconditions wherein the suppression of the immunosuppressive effects ofVISTA on T cell immunity is desired such as cancer and infection.However, to the best of the inventors' knowledge no anti-human VISTAantibody or antibody fragment has been previously identified whichagonizes the effects of human VISTA. Such agonistic anti-human VISTAantibodies and antibody fragments would be desirable in treatingconditions wherein the suppression of immunity, particularly T cellimmunity is desired and/or conditions wherein VISTA expression isaberrantly downregulated.

SUMMARY

It is an object of the invention to provide novel antibodies andantibody fragments which specifically bind to human VISTA and variantsthereof, e.g., chimeric, human, humanized or multispecific anti-humanVISTA antibodies which specifically bind to human VISTA and whichpromote or mimic the effects of human VISTA on immunity.

It is a specific object of the invention to provide agonistic antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human VISTA wherein the agonistic antibody orantibody fragment binds to the same or overlapping epitope as any one ofthe anti-human VISTA antibodies having the CDR and variable heavy andlight polypeptides shown in FIG. 4A-4JJ.

It is a specific object of the invention to provide an isolated antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human V-domain Ig Suppressor of T cell Activation(human VISTA), wherein the antibody or antibody fragment agonizes orpromotes one or more of the effects of VISTA on immunity, e.g.,comprising a human IgG2 constant or human IgG2 Fc region optionallywherein the human IgG2 constant or Fc region binds to Fc gamma receptorsincluding human CD32A and/or containing a human IgG2 constant or Fcregion which comprises the native human IgG2 binding to Fc gammareceptors and/or an IgG2 which binds to FcyRs including one or more ofhFcyRI (CD64), FcyRIIA or hFcyRIIB, (CD32 or CD32A) and FcyRIIIA (CD16A)or FcyRIIB (CD16B).

It is a specific object of the invention to provide agonistic antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human VISTA wherein the agonistic antibody orantibody fragment binds to a VISTA epitope which includes or overlapswith the epitope bound by any of the anti-human VISTA antibodies havingthe sequences of FIG. 4A-4JJ.

It is a specific object of the invention to provide agonistic antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human VISTA wherein the agonistic antibody orantibody fragment binds or interacts with one of more residues of anepitope comprising residues of LLDSGLYCCLVVEIRHHHSEHRVH.

It is a specific object of the invention to provide agonistic antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human VISTA wherein the agonistic antibody orantibody fragment binds or interacts with one of more residues of anepitope comprising one or more residues of 79EVQTCSERRPIR90,48NVTLTCRLLGPV60, 153HHHSEHRVHGAM164, 52LTCRLLGPV60,56LLGPVDKGHDVTFYK70, 113LAQRHGLESASDHHG127, 153HHHSEHRVHGAM164,93TFQDLHLHHGGHQAA107, 146CLVVEIRHHHSEH158, 53TCRLLGPVDKG63, 123SDHHG127and/or 153HHHSEHRVHGAM164.

It is a specific object of the invention to provide agonistic antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human VISTA wherein the agonistic antibody orantibody fragment binds or interacts with one of more residues of anepitope comprising one or more residues of 79EVQTCSERRPIR90.

It is a specific object of the invention to provide agonistic antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human VISTA wherein the agonistic antibody orantibody fragment promotes or enhances at least one effect of humanVISTA on immunity, e.g. its suppressive effect on any one or more of Tcell immunity, activation of monocytes, induction of T-cellproliferation; induction or suppression of cytokine expression,increased survival of monocytes, induction of antibody-dependentcell-mediated cytotoxicity (ADCC) in cells-expressing VISTA; andinduction of antibody-dependent cellular phagocytosis (ADCP) incells-expressing VISTA.

It is a specific object of the invention to provide agonistic antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human VISTA wherein the agonistic antibody orantibody fragment comprising an antigen binding region that specificallybinds to human VISTA, wherein the antibody or antibody fragment whichcomprises variable heavy and light sequences having the identical CDRpolypeptides as any one of the anti-human VISTA antibodies having theCDR and variable heavy and light polypeptides shown in FIG. 4A-4JJ, withthe proviso that if said antibody or fragment comprises an antagonistanti-human VISTA antibody or antibody fragment then the antibody orantibody fragment does not comprise the same CDRs as any one of VSTB112,VSTB116, VSTB95, VSTB50, VSTB53 or VSTB60.

It is a specific object of the invention to provide agonistic antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human VISTA wherein the agonistic antibody orantibody which comprises an antagonist anti-human VISTA antibody orfragment then the antibody or antibody fragment does not comprise thesame CDRs as any one of VSTB112, VSTB116, VSTB95, VSTB50, VSTB53 orVSTB60.

It is a specific object of the invention to provide agonistic antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human VISTA wherein the agonistic antibody orantibody fragment comprises a variable heavy and/or variable lightpolypeptide having at least 90% sequence identity to those of ananti-human VISTA antibody selected from any one of VSTB49-VSTB116,wherein the variable heavy and light polypeptide sequences thereof areshown in FIG. 4A-4JJ, with the proviso that if said antibody or fragmentcomprises an antagonist anti-human VISTA antibody or fragment then theantibody or antibody fragment does not comprise the same CDRs as any oneof VSTB112, VSTB116, VSTB95, VSTB50, VSTB53 or VSTB60.

It is a specific object of the invention to provide agonistic antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human VISTA wherein the agonistic antibody orantibody comprises an antigen binding region that specifically binds tohuman VISTA wherein the agonistic antibody or antibody fragmentcomprises a variable heavy and/or variable light polypeptide having atleast 95% sequence identity to those of an anti-human VISTA antibodyselected from any one of VSTB49-VSTB116, wherein the variable heavy andlight polypeptide sequences thereof are shown in FIG. 4A-4JJ, with theproviso that if said antibody or fragment comprises an antagonistanti-human VISTA antibody or antibody fragment then the antibody orantibody fragment does not comprise the same CDRs as any one of VSTB112,VSTB116, VSTB95, VSTB50, VSTB53 or VSTB60.

It is a specific object of the invention to provide agonistic antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human VISTA wherein the agonistic antibody orantibody fragment comprises a variable heavy and/or variable lightpolypeptide having at least 96-99% sequence identity to those of ananti-human VISTA antibody selected from any one of VSTB49-VSTB116,wherein the variable heavy and light polypeptide sequences thereof areshown in FIG. 4A-4JJ, with the proviso that if said antibody or fragmentcomprises an antagonist anti-human VISTA antibody or antibody fragmentthen the antibody or antibody fragment does not comprise the same CDRsas any one of VSTB112, VSTB116, VSTB95, VSTB50, VSTB53 or VSTB60.

It is a specific object of the invention to provide agonistic antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human VISTA wherein the agonistic antibody orantibody fragment which comprises a variable heavy and/or variable lightpolypeptide identical to those of an anti-human VISTA antibody selectedfrom one of VSTB49-VSTB116, wherein the variable heavy and lightpolypeptide sequences thereof are shown in FIG. 4A-4JJ, with the provisothat if said antibody or fragment comprises an antagonist anti-humanVISTA antibody or antibody fragment then the antibody or antibodyfragment does not comprise the same CDRs as any one of VSTB112, VSTB116,VSTB95, VSTB50, VSTB53 or VSTB60.

It is a specific object of the invention to provide an antagonisticantibody or antibody fragment thereof comprising an antigen bindingregion that specifically binds to human VISTA according to any of theforegoing which antagonizes or blocks at least one effect of human VISTAon immunity.

It is a specific object of the invention to provide an agonisticantibody or antibody fragment thereof comprising an antigen bindingregion that specifically binds to human VISTA according to any of theforegoing which agonizes or promotes at least one effect of human VISTAon immunity.

It is a specific object of the invention to provide an agonisticantibody or antibody fragment thereof comprising an antigen bindingregion that specifically binds to human VISTA according to any of theforegoing which comprises a human constant domain.

It is a specific object of the invention to provide agonistic antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human VISTA according to any of the foregoingwhich comprises a human constant domain selected from IgGI, IgG2, IgG3and IgG4, which optionally is modified, e.g., by deletion, substitutionor addition mutations or any combination of the foregoing.

It is a specific object of the invention to provide agonistic antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human VISTA according to any of the foregoingwherein the antibody fragment comprises or is a Fab, F(ab′)2, or scFvantibody fragment.

It is a specific object of the invention to provide an antagonisticantibody or antibody fragment thereof comprising an antigen bindingregion that specifically binds to human VISTA according to any of theforegoing which blocks or suppresses at least one of the effects ofhuman VISTA on immunity, e.g., selected from its suppressive effect on Tcell immunity, activation of monocytes, or T-cell proliferation;induction or suppression of cytokine expression, increased survival ofmonocytes, suppression of antibody-dependent cell-mediated cytotoxicity(ADCC) of cells-expressing VISTA; and suppression of antibody-dependentcellular phagocytosis (ADCP) of cells-expressing VISTA.

It is a specific object of the invention to provide agonistic antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human VISTA according to any of the foregoingwhich promotes or enhances at least one of the effects of human VISTA onimmunity, e.g., selected from its suppressive effect T cell immunity,activation of monocytes, suppression of T-cell proliferation; inductionor suppression of cytokine expression, increased survival of monocytes,suppression of antibody-dependent cell-mediated cytotoxicity (ADCC) incells-expressing VISTA; and suppression of antibody-dependent cellularphagocytosis (ADCP) of cells-expressing VISTA.

It is a specific object of the invention to provide agonistic antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human VISTA according to any of the foregoingwhich comprises a human IgG2 constant or Fc region.

It is a specific object of the invention to provide agonistic antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human VISTA according to any of the foregoing thatpromotes or enhances the suppressive effect of human VISTA on immunity,e.g. its effect on any one or more of T cell immunity, activation ofmonocytes, T-cell proliferation; cytokine expression, survival ofmonocytes, antibody-dependent cell-mediated cytotoxicity (ADCC) incells-expressing VISTA; and antibody-dependent cellular phagocytosis(ADCP) in cells-expressing VISTA.

It is a specific object of the invention to provide an agonisticantibody or antibody fragment thereof comprising an antigen bindingregion that specifically binds to human VISTA according to any of theforegoing which inhibits T cell immunity and/or proinflammatory cytokineexpression.

It is a specific object of the invention to provide an agonisticantibody or antibody fragment thereof comprising an antigen bindingregion that specifically binds to human VISTA according to any of theforegoing which is a human, humanized or chimeric antibody thatcomprises a human Fc region, e.g., human IgGI, IgG2, IgG3 and IgG4 or achimera of any of the foregoing.

It is a specific object of the invention to provide an agonisticantibody or antibody fragment thereof comprising an antigen bindingregion that specifically binds to human VISTA according to any of theforegoing which is chimeric, human or humanized.

It is a specific object of the invention to provide agonistic antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human VISTA according to any of the foregoingwhich comprises a human IgG2 constant domain or Fc region whichpotentially may be mutated.

It is a specific object of the invention to provide an agonisticantibody or antibody fragment thereof comprising an antigen bindingregion that specifically binds to human VISTA according to any of theforegoing which comprises a human IgG2 constant domain or fragmentthereof or an hIgGI, hIgG3, hIgG4, IgA, IgD, IgE, or IgM, wherein theentire or substantially the entire hinge and CHI domains of saidantibody and optionally the entire or substantially the entire lightchain constant region have been replaced with the corresponding entireor substantially the entire light chain, and the hinge and CHI domains(“H2 regions” or “H2 domains”) of hIgG2.

It is a specific object of the invention to provide agonistic antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human VISTA according to any of the foregoingwhich (i) comprises an IgG2 Fc region wherein either or both of theheavy chain cysteine residue at position 127 and the light chaincysteine residue at position 214 (wherein numbering is according toKabat) are deleted or changed to a different amino acid residue,resulting in an increase in the agonistic properties of the resultantmodified antibody relative to an antibody wherein these residues areunchanged, (ii) the cysteine residue at position 214 in the H2 region ofsaid antibody is mutated or substituted with another amino acid and/orone or more of the cysteine residues at positions 127, 232 or 233 of theheavy chain are deleted or substituted with another amino acid, (iii) itcomprises a human IgG2 constant domain wherein at least one cysteineresidue is deleted or changed to another amino acid, (iv) it competeswith or binds to the same epitope on human VISTA as VSTB95 (variableheavy and light sequences shown in FIG. 4A-4JJ).

It is a specific object of the invention to provide agonistic antibodyor antibody fragment thereof comprising an antigen binding region thatspecifically binds to human VISTA according to any of the foregoingwhich:

-   -   (i) comprises the V_(H) CDRs of SEQ ID NO: 100, 101 and 102 and        the V_(L) CDRs of SEQ ID NO: 103, 104 and 105;    -   (ii) comprises the V_(H) CDRs of SEQ ID NO: 110, 111 and 112 and        the V_(L) CDRs of SEQ ID NO:113, 114 and 115;    -   (iii) comprises the V_(H) CDRs of SEQ ID NO: 120, 121 and 122        and the V_(L) CDRs of SEQ ID NO: 123, 124 and 125;    -   (iv) comprises the V_(H) CDRs of SEQ ID NO: 130, 131 and 132 and        the V_(L) CDRs of SEQ ID NO: 133, 134 and 135;    -   (v) comprises the V_(H) CDRs of SEQ ID NO: 140, 141 and 142 and        the V_(L) CDRs of SEQ ID NO:143, 144 and 145;    -   (vi) comprises the V_(H) CDRs of SEQ ID NO: 150, 151 and 152 and        the V_(L) CDRs of SEQ ID NO:153, 154 and 155;    -   (vii) comprises the V_(H) CDRs of SEQ ID NO: 160, 161 and 162        and the V_(L) CDRs of SEQ ID NO:163, 164 and 165;    -   (viii) comprises the V_(H) CDRs of SEQ ID NO: 170, 171 and 172        and the V_(L) CDRs of SEQ ID NO:173, 174 and 175;    -   (ix) comprises the V_(H) CDRs of SEQ ID NO: 180, 181 and 182 and        the V_(L) CDRs of SEQ ID NO:183, 184 and 185;    -   (x) comprises the V_(H) CDRs of SEQ ID NO: 190, 191 and 192 and        the V_(L) CDRs of SEQ ID NO:193, 194 and 195;    -   (xi) comprises the V_(H) CDRs of SEQ ID NO:200, 201 and 202 and        the V_(L) CDRs of SEQ ID NO:203, 204 and 205;    -   (xii) comprises the V_(H) CDRs of SEQ ID NO:210, 211 and 212 and        the V_(L) CDRs of SEQ ID NO:213, 214 and 215;    -   (xiii) comprises the V_(H) CDRs of SEQ ID NO:220, 221 and 222        and the V_(L) CDRs of SEQ ID NO:223, 224 and 225;    -   (xiv) comprises the V_(H) CDRs of SEQ ID NO:230, 231 and 232 and        the V_(L) CDRs of SEQ ID NO:233, 234 and 235;    -   (xv) comprises the V_(H) CDRs of SEQ ID NO:240, 241 and 242 and        the V_(L) CDRs of SEQ ID NO:243, 244 and 245;    -   (xvi) comprises the V_(H) CDRs of SEQ ID NO:250, 251 and 252 and        the V_(L) CDRs of SEQ ID NO:253, 254 and 255;    -   (xvii) comprises the V_(H) CDRs of SEQ ID NO:260, 261 and 262        and the V_(L) CDRs of SEQ ID NO:263, 264 and 265;    -   (xviii) comprises the V_(H) CDRs of SEQ ID NO:270, 271 and 272        and the V_(L) CDRs of SEQ ID NO:273, 274 and 275;    -   (xix) comprises the V_(H) CDRs of SEQ ID NO:280, 281 and 282 and        the V_(L) CDRs of SEQ ID NO:283, 284 and 285;    -   (xx) comprises the V_(H) CDRs of SEQ ID NO:290, 291 and 292 and        the V_(L) CDRs of SEQ ID NO:293, 294 and 295;    -   (xxi) comprises the V_(H) CDRs of SEQ ID NO:300, 301 and 302 and        the V_(L) CDRs of SEQ ID NO:303, 304 and 305;    -   (xxii) comprises the V_(H) CDRs of SEQ ID NO:310, 311 and 312        and the V_(L) CDRs of SEQ ID NO:313, 314 and 315;    -   (xxiii) comprises the V_(H) CDRs of SEQ ID NO:320, 321 and 322        and the V_(L) CDRs of SEQ ID NO:323, 324 and 325;    -   (xxiv) comprises the V_(H) CDRs of SEQ ID NO:330, 331 and 332        and the V_(L) CDRs of SEQ ID NO:333, 334 and 335;    -   (xxv) comprises the V_(H) CDRs of SEQ ID NO:340, 341 and 342 and        the V_(L) CDRs of SEQ ID NO:343, 344 and 345;    -   (xxvi) comprises the V_(H) CDRs of SEQ ID NO:350, 351 and 352        and the V_(L) CDRs of SEQ ID NO:353, 354 and 355;    -   (xxvii) comprises the V_(H) CDRs of SEQ ID NO:360, 361 and 362        and the V_(L) CDRs of SEQ ID NO:363, 364 and 365;    -   (xxviii) comprises the V_(H) CDRs of SEQ ID NO:370, 371 and 372        and the V_(L) CDRs of SEQ ID NO:373, 374 and 375;    -   (xxix) comprises the V_(H) CDRs of SEQ ID NO:380, 381 and 382        and the V_(L) CDRs of SEQ ID NO:383, 384 and 385;    -   (xxx) comprises the V_(H) CDRs of SEQ ID NO:390, 391 and 392 and        the V_(L) CDRs of SEQ ID NO:393, 394 and 395;    -   (xxxi) comprises the V_(H) CDRs of SEQ ID NO:400, 401 and 402        and the V_(L) CDRs of SEQ ID NO:403, 404 and 405;    -   (xxxii) comprises the V_(H) CDRs of SEQ ID NO:410, 411 and 412        and the V_(L) CDRs of SEQ ID NO:413, 414 and 415;    -   (xxxiii) comprises the V_(H) CDRs of SEQ ID NO:420, 421 and 422        and the V_(L) CDRs of SEQ ID NO:423, 424 and 425;    -   (xxxiv) comprises the V_(H) CDRs of SEQ ID NO:430, 431 and 432        and the V_(L) CDRs of SEQ ID NO:433, 434 and 435;    -   (xxxv) comprises the V_(H) CDRs of SEQ ID NO:440, 441 and 442        and the V_(L) CDRs of SEQ ID NO:443, 444 and 445;    -   (xxxvi) comprises the V_(H) CDRs of SEQ ID NO:450, 451 and 452        and the V_(L) CDRs of SEQ ID NO:453, 454 and 455;    -   (xxxvii) comprises the V_(H) CDRs of SEQ ID NO:460, 461 and 462        and the V_(L) CDRs of SEQ ID NO:463, 464 and 465;    -   (xxxviii) comprises the V_(H) CDRs of SEQ ID NO:470, 471 and 472        and the V_(L) CDRs of SEQ ID NO:473, 474 and 475;    -   (xxxix) comprises the V_(H) CDRs of SEQ ID NO:480, 481 and 482        and the V_(L) CDRs of SEQ ID NO:483, 484 and 485;    -   (xl) comprises the V_(H) CDRs of SEQ ID NO:490, 491 and 492 and        the VL CDR polypeptides of SEQ ID NO:493, 494 and 495;    -   (xli) comprises the V_(H) CDRs of SEQ ID NO:500, 501 and 502 and        the VL CDR polypeptides of SEQ ID NO:503, 504 and 505;    -   (xlii) comprises the V_(H) CDRs of SEQ ID NO:510, 511 and 512        and the VL CDR polypeptides of SEQ ID NO:513, 514 and 515;    -   (xliii) comprises the V_(H) CDRs of SEQ ID NO:520, 521 and 522        and the VL CDR polypeptides of SEQ ID NO:523, 524 and 525;    -   (xliv) comprises the V_(H) CDRs of SEQ ID NO:530, 531 and 532        and the VL CDR polypeptides of SEQ ID NO:533, 534 and 535;    -   (xlv) comprises the V_(H) CDRs of SEQ ID NO:540, 541 and 542 and        the VL CDR polypeptides of SEQ ID NO:543, 544 and 545;    -   (xlvi) comprises the V_(H) CDRs of SEQ ID NO:550, 551 and 552        and the VL CDR polypeptides of SEQ ID NO:553, 554 and 555;    -   (xlvii) comprises the V_(H) CDRs of SEQ ID NO:560, 561 and 562        and the V_(L) CDRs of SEQ ID NO:563, 564 and 565;    -   (xlviii) comprises the V_(H) CDRs of SEQ ID NO:570, 571 and 572        and the V_(L) CDRs of SEQ ID NO:573, 574 and 575;    -   (xlix) comprises the V_(H) CDRs of SEQ ID NO:580, 581 and 582        and the V_(L) CDRs of SEQ ID NO:583, 584 and 585;    -   (l) comprises the V_(H) CDRs of SEQ ID NO:590, 591 and 592 and        the V_(L) CDRs of SEQ ID NO:593, 594 and 595;    -   (li) comprises the V_(H) CDRs of SEQ ID NO:600, 601 and 602 and        the V_(L) CDRs of SEQ ID NO:603, 604 and 605;    -   (lii) comprises the V_(H) CDRs of SEQ ID NO:610, 611 and 612 and        the V_(L) CDRs of SEQ ID NO:613, 614 and 615;    -   (liii) comprises the V_(H) CDRs of SEQ ID NO:620, 621 and 622        and the V_(L) CDRs of SEQ ID NO:623, 624 and 625;    -   (liv) comprises the V_(H) CDRs of SEQ ID NO:630, 631 and 632 and        the V_(L) CDRs of SEQ ID NO:633, 634 and 635;    -   (lv) comprises the V_(H) CDRs of SEQ ID NO:640, 641 and 642 and        the V_(L) CDRs of SEQ ID NO:643, 644 and 645;    -   (lvi) comprises the V_(H) CDRs of SEQ ID NO:650, 651 and 652 and        the V_(L) CDRs of SEQ ID NO:653, 654 and 655;    -   (lvii) comprises the V_(H) CDRs of SEQ ID NO:660, 661 and 662        and the V_(L) CDRs of SEQ ID NO:663, 664 and 665;    -   (lviii) comprises the V_(H) CDRs of SEQ ID NO:670, 671 and 672        and the V_(L) CDRs of SEQ ID NO:673, 674 and 675;    -   (lix) comprises the V_(H) CDRs of SEQ ID NO:680, 681 and 682 and        the V_(L) CDRs of SEQ ID NO:683, 684 and 685;    -   (lx) comprises the V_(H) CDRs of SEQ ID NO:690, 691 and 692 and        the V_(L) CDRs of SEQ ID NO:693, 694 and 695;    -   (lxi) comprises the V_(H) CDRs of SEQ ID NO:700, 701 and 702 and        the V_(L) CDRs of SEQ ID NO:703, 704 and 705;    -   (lxii) comprises the V_(H) CDRs of SEQ ID NO:710, 711 and 712        and the V_(L) CDRs of SEQ ID NO:713, 714 and 715;    -   (lxiii) comprises the V_(H) CDRs of SEQ ID NO:720, 721 and 722        and the V_(L) CDRs of SEQ ID NO:723, 724 and 725;    -   (lxiv) comprises the V_(H) CDRs of SEQ ID NO:730, 731 and 732        and the V_(L) CDRs of SEQ ID NO:733, 734 and 735;    -   (lxv) comprises the V_(H) CDRs of SEQ ID NO:740, 741 and 742 and        the V_(L) CDRs of SEQ ID NO:743, 744 and 745;    -   (lxvi) comprises the V_(H) CDRs of SEQ ID NO:750, 751 and 752        and the V_(L) CDRs of SEQ ID NO:753, 754 and 755;    -   (lxvii) comprises the V_(H) CDRs of SEQ ID NO:760, 761 and 762        and the V_(L) CDRs of SEQ ID NO:763, 764 and 765;    -   (lxviii) comprises the V_(H) CDRs of SEQ ID NO:770, 771 and 772        and the V_(L) CDRs of SEQ ID NO:773, 774 and 775;    -   (lxix) comprises the V_(H) CDRs of SEQ ID NO:780, 781 and 782        and the V_(L) CDRs of SEQ ID NO:783, 784 and 785;    -   (lxx) comprises the V_(H) CDRs of SEQ ID NO:790, 791 and 792 and        the V_(L) CDRs of SEQ ID NO:793, 794 and 795;    -   (lxxi) comprises the V_(H) CDRs of SEQ ID NO:800, 801 and 802        and the V_(L) CDRs of SEQ ID NO:803, 804 and 805;    -   (lxxii) comprises the V_(H) CDRs of SEQ ID NO:810, 811 and 812        and the V_(L) CDRs of SEQ ID NO: 813, 814 and 815.

It is an object of the invention to provide a VISTA agonist according toany of the foregoing which:

-   -   (i) comprises the V_(H) polypeptide of SEQ ID NO: 106 and the        V_(L) polypeptide of SEQ ID NO: 108;    -   (ii) comprises the V_(H) polypeptide of SEQ ID NO: 116 and the        V_(L) polypeptide of SEQ ID NO: 118;    -   (iii) comprises the V_(H) polypeptide of SEQ ID NO: 126 and the        V_(L) polypeptide of SEQ ID NO: 128;    -   (iv) comprises the V_(H) polypeptide of SEQ ID NO: 136 and the        V_(L) polypeptide f SEQ ID NO: 138;    -   (v) comprises the V_(H) polypeptide of SEQ ID NO: 146 and the        V_(L) polypeptide of SEQ ID NO: 148;    -   (vi) comprises the V_(H) polypeptide of SEQ ID NO: 156 and the        V_(L) polypeptide of SEQ ID NO: 158;    -   (vii) comprises the V_(H) polypeptide of SEQ ID NO: 166 and the        V_(L) polypeptide of SEQ ID NO: 168;    -   (viii) comprises the V_(H) polypeptide of SEQ ID NO: 176 and the        V_(L) polypeptide of SEQ ID NO: 178;    -   (ix) comprises the V_(H) polypeptide of SEQ ID NO: 186 and the        V_(L) polypeptide of SEQ ID NO: 188;    -   (x) comprises the V_(H) polypeptide of SEQ ID NO: 196 and the        V_(L) polypeptide of SEQ ID NO: 198;    -   (xi) comprises the V_(H) polypeptide of SEQ ID NO:206 and the        V_(L) polypeptide of SEQ ID NO:208;    -   (xii) comprises the V_(H) polypeptide of SEQ ID NO:216 and the        V_(L) polypeptide of SEQ ID NO:218;    -   (xiii) comprises the V_(H) polypeptide of SEQ ID NO:226 and the        V_(L) polypeptide of SEQ ID NO:228;    -   (xiv) comprises the V_(H) polypeptide of SEQ ID NO:236 and the        V_(L) polypeptide of SEQ ID NO:238;    -   (xv) comprises the V_(H) polypeptide of SEQ ID NO:246 and the        V_(L) polypeptide of SEQ ID NO:248;    -   (xvi) comprises the V_(H) polypeptide of SEQ ID NO:256 and the        V_(L) polypeptide of SEQ ID NO:258;    -   (xvii) comprises the V_(H) polypeptide of SEQ ID NO:266 and the        V_(L) polypeptide of SEQ ID NO:268;    -   (xviii) comprises the V_(H) polypeptide of SEQ ID NO:276 and the        V_(L) polypeptide of SEQ ID NO:278;    -   (xix) comprises the V_(H) polypeptide of SEQ ID NO:286 and the        V_(L) polypeptide of SEQ ID NO:288;    -   (xx) comprises the V_(H) polypeptide of SEQ ID NO:296 and the        V_(L) polypeptide of SEQ ID NO:298;    -   (xxi) comprises the V_(H) polypeptide of SEQ ID NO:306 and the        V_(L) polypeptide of SEQ ID NO:308;    -   (xxii) comprises the V_(H) polypeptide of SEQ ID NO:316 and the        V_(L) polypeptide of SEQ ID NO:318;    -   (xxiii) comprises the V_(H) polypeptide of SEQ ID NO:326 and the        V_(L) polypeptide of SEQ ID NO:328;    -   (xxiv) comprises the V_(H) polypeptide of SEQ ID NO:336 and the        V_(L) polypeptide of SEQ ID NO:338;    -   (xxv) comprises the V_(H) polypeptide of SEQ ID NO:346 and the        V_(L) polypeptide of SEQ ID NO:348;    -   (xxvi) comprises the V_(H) polypeptide of SEQ ID NO:356 and the        V_(L) polypeptide of SEQ ID NO:358;    -   (xxvii) comprises the V_(H) polypeptide of SEQ ID NO:366 and the        V_(L) polypeptide of SEQ ID NO:368;    -   (xxviii) comprises the V_(H) polypeptide of SEQ ID NO:376 and        the V_(L) polypeptide of SEQ ID NO:378;    -   (xxix) comprises the V_(H) polypeptide of SEQ ID NO:386 and the        V_(L) polypeptide of SEQ ID NO:388;    -   (xxx) comprises the V_(H) polypeptide of SEQ ID NO:396 and the        V_(L) polypeptide of SEQ ID NO:398;    -   (xxxi) comprises the V_(H) polypeptide of SEQ ID NO:406 and the        V_(L) polypeptide of SEQ ID NO:408;    -   (xxxii) comprises the V_(H) polypeptide of SEQ ID NO:416 and the        V_(L) polypeptide of SEQ ID NO:418;    -   (xxxiii) comprises the V_(H) polypeptide of SEQ ID NO:426 and        the V_(L) polypeptide of SEQ ID NO:428;    -   (xxxiv) comprises the V_(H) polypeptide of SEQ ID NO:436 and the        V_(L) polypeptide of SEQ ID NO:438;    -   (xxxv) comprises the V_(H) polypeptide of SEQ ID NO:446 and the        V_(L) polypeptide of SEQ ID NO:448;    -   (xxxvi) comprises the V_(H) polypeptide of SEQ ID NO:456 and the        V_(L) polypeptide of SEQ ID NO:458;    -   (xxxvii) comprises the V_(H) polypeptide of SEQ ID NO:466 and        the V_(L) polypeptide of SEQ ID NO:468;    -   (xxxviii) comprises the V_(H) polypeptide of SEQ ID NO:476 and        the V_(L) polypeptide of SEQ ID NO:478;    -   (xxxix) comprises the V_(H) polypeptide of SEQ ID NO:486 and the        V_(L) polypeptide of SEQ ID NO:488;    -   (xl) comprises the V_(H) polypeptide of SEQ ID NO:496 and the        V_(L) polypeptide of SEQ ID NO:498;    -   (xli) comprises the V_(H) polypeptide of SEQ ID NO:506 and the        V_(L) polypeptide of SEQ ID NO:508;    -   (xlii) comprises the V_(H) polypeptide of SEQ ID NO:516 and the        V_(L) polypeptide of SEQ ID NO:518;    -   (xliii) comprises the V_(H) polypeptide of SEQ ID NO:526 and the        V_(L) polypeptide of SEQ ID NO:528;    -   (xliv) comprises the V_(H) polypeptide of SEQ ID NO:536 and the        V_(L) polypeptide of SEQ ID NO:533, 534 and 535;    -   (xlv) comprises the V_(H) polypeptide of SEQ ID NO:546 and the        V_(L) polypeptide of SEQ ID NO:548;    -   (xlvi) comprises the V_(H) polypeptide of SEQ ID NO:556 and the        V_(L) polypeptide of SEQ ID NO:558;    -   (xlvii) comprises the V_(H) polypeptide of SEQ ID NO:566 and the        V_(L) polypeptide of SEQ ID NO:568;    -   (xlviii) comprises the V_(H) polypeptide of SEQ ID NO:576 and        the V_(L) polypeptide of SEQ ID NO:578;    -   (xlix) comprises the V_(H) polypeptide of SEQ ID NO:586 and the        V_(L) polypeptide of SEQ ID NO:588;    -   (l) comprises the V_(H) polypeptide of SEQ ID NO:596 and the        V_(L) polypeptide of SEQ ID NO:598;    -   (li) comprises the V_(H) polypeptide of SEQ ID NO:606 and the        V_(L) polypeptide of SEQ ID NO:608;    -   (lii) comprises the V_(H) polypeptide of SEQ ID NO:616 and the        V_(L) polypeptide of SEQ ID NO:618;    -   (liii) comprises the V_(H) polypeptide of SEQ ID NO:626 and the        V_(L) polypeptide of SEQ ID NO:628;    -   (liv) comprises the V_(H) polypeptide of SEQ ID NO:636 and the        V_(L) polypeptide of SEQ ID NO:638;    -   (lv) comprises the V_(H) polypeptide of SEQ ID NO:646 and the        V_(L) polypeptide of SEQ ID NO:648;    -   (lvi) comprises the V_(H) polypeptide of SEQ ID NO:656 and the        V_(L) polypeptide of SEQ ID NO:658;    -   (lvii) comprises the V_(H) polypeptide of SEQ ID NO:666 and the        V_(L) polypeptide of SEQ ID NO:668;    -   (lviii) comprises the V_(H) polypeptide of SEQ ID NO:676 and the        V_(L) polypeptide of SEQ ID NO:678;    -   (lix) comprises the V_(H) polypeptide of SEQ ID NO:686 and the        V_(L) polypeptide of SEQ ID NO:688;    -   (lx) comprises the V_(H) polypeptide of SEQ ID NO:696 and the        V_(L) polypeptide of SEQ ID NO:698;    -   (lxi) comprises the V_(H) polypeptide of SEQ ID NO:706 and the        V_(L) polypeptide of SEQ ID NO:708;    -   (lxii) comprises the V_(H) polypeptide of SEQ ID NO:716 and the        V_(L) polypeptide of SEQ ID NO:718;    -   (lxiii) comprises the V_(H) polypeptide of SEQ ID NO:726 and the        V_(L) polypeptide of SEQ ID NO:728;    -   (lxiv) comprises the V_(H) polypeptide of SEQ ID NO:736 and the        V_(L) polypeptide of SEQ ID NO:738;    -   (lxv) comprises the V_(H) polypeptide of SEQ ID NO:746 and the        V_(L) polypeptide of SEQ ID NO:748;    -   (lxvi) comprises the V_(H) polypeptide of SEQ ID NO:756 and the        V_(L) polypeptide of SEQ ID NO:758;    -   (lxvii) comprises the V_(H) polypeptide of SEQ ID NO:766 and the        V_(L) polypeptide of SEQ ID NO:768;    -   (lxviii) comprises the V_(H) polypeptide of SEQ ID NO:776 and        the V_(L) polypeptide of SEQ ID NO:778;    -   (lxix) comprises the V_(H) polypeptide of SEQ ID NO:786 and the        V_(L) polypeptide of SEQ ID NO:788;    -   (lxx) comprises the V_(H) polypeptide of SEQ ID NO:796 and the        V_(L) polypeptide of SEQ ID NO:798;    -   (lxxi) comprises the V_(H) polypeptide of SEQ ID NO:806 and the        V_(L) polypeptide of SEQ ID NO:808; and    -   (lxxii) comprises the V_(H) polypeptide of SEQ ID NO:816 and the        V_(L) polypeptide of SEQ ID NO: 818.

It is a specific object of the invention to provide an agonisticantibody or antibody fragment thereof comprising an antigen bindingregion that specifically binds to human VISTA according to any of theforegoing which comprises a human IgG2 constant domain whereinoptionally at least one cysteine residue is deleted or changed toanother amino acid.

It is a specific object of the invention to provide an agonisticantibody or antibody fragment thereof comprising an antigen bindingregion that specifically binds to human VISTA according to any of theforegoing which mediates any one or combination of at least one of thefollowing immunoinhibitory effects: (i) decreases immune response, (ii)decreases T cell activation, (iii) decreases cytotoxic T cell activity,(iv) decreases natural killer (NK) cell activity, (v) decreases T-cellactivity, (vi) decreases pro-inflammatory cytokine secretion, (vii)decreases IL-2 secretion; (viii) decreases interferon-γ production, (ix)decreases ThI response, (x) decreases Th2 response, (xi) increases cellnumber and/or activity of regulatory T cells, (xii) increases regulatorycell activity and/or one or more of myeloid derived suppressor cells(MDSCs), iMCs, mesenchymal stromal cells, TIE2-expressing monocytes,(xiii) increases regulatory cell activity and/or the activity of one ormore of myeloid derived suppressor cells (MDSCs), iMCs, mesenchymalstromal cells, TIE2-expressing monocytes, (xiii) increases M2macrophages, (xiv) increases M2 macrophage activity, (xv) increases N2neutrophils, (xvi) increases N2 neutrophils activity, (xvii) increasesinhibition of T cell activation, (xviii) increases inhibition of CTLactivation, (xix) increases inhibition of NK cell activation, (xx)increases T cell exhaustion, (xxi) decreases T cell response, (xxii)decreases activity of cytotoxic cells, (xxiii) reduces antigen-specificmemory responses, (xxiv) inhibits apoptosis or lysis of cells, (xxv)decreases cytotoxic or cytostatic effect on cells, (xxvi) reduces directkilling of cells, (xxvii) decreases ThI7 activity, and/or (xxviii)reduces complement dependent cytotoxicity and/or antibody dependentcell-mediated cytotoxicity, with the proviso that said anti-VISTAantibody or antigen-binding fragment may elicit an opposite effect toone or more of (i)-(xxviii) and optionally is used to treatautoimmunity, allergy, inflammation, transplant or sepsis.

It is a specific object of the invention to provide a pharmaceutical ordiagnostic composition comprising an agonistic antibody or antibodyfragment thereof comprising an antigen binding region that specificallybinds to human VISTA according to any of the foregoing.

It is a specific object of the invention to provide a method oftreatment and/or diagnosis, or use of a composition containing at leastone antagonistic antibody or antibody fragment according to any of theforegoing claims for diagnostic or therapeutic use, which method or usecomprises the administration to a subject in need thereof at least onedosage or composition comprising a therapeutically or diagnosticallyeffective amount of at least one at least one antagonistic antibody orantibody fragment according to any of the foregoing, e.g., cancer or aninfectious disorder, optionally wherein the cancer is a blood cancer orsolid tumor, e.g., one surrounded by a tumor stroma comprising myeloidcells, T-cells, or a combination of myeloid cells and T-cells or acancer selected from leukemia, lymphoma, myelodysplastic syndrome ormyeloma, lung cancer or a combination thereof or a leukemia whichcomprises acute lymphoblastic leukemia (ALL), chronic lymphocyticleukemia (CLL), acute myeloid (myelogenous) leukemia (AML), chronicmyelogenous leukemia (CML); hairy cell leukemia, T-cell prolymphocyticleukemia, large granular lymphocytic leukemia, or adult T-cell leukemia.

It is a specific object of the invention to provide a method oftreatment and/or diagnosis, or use of a composition containing at leastone agonistic antibody or antibody fragment according to any of theforegoing claims for diagnostic or therapeutic use, which method or usecomprises the administration to a subject in need thereof at least onedosage or composition comprising a therapeutically or diagnosticallyeffective amount of at least one at least one agonistic antibody orantibody fragment according to any of the foregoing or compositioncontaining according to any of the foregoing.

It is a specific object of the invention to provide a method or use ofany agonistic antibody or antibody fragment according to any of theforegoing for effecting in vitro and/or in vivo any one or combinationof at least one of the following immunoinhibitory effects: (i) decreasesimmune response, (ii) decreases T cell activation, (iii) decreasescytotoxic T cell activity, (iv) decreases natural killer (NK) cellactivity, (v) decreases T-cell activity, (vi) decreases pro-inflammatorycytokine secretion, (vii) decreases IL-2 secretion; (viii) decreasesinterferon-γ production, (ix) decreases ThI response, (x) decreases Th2response, (xi) increases cell number and/or activity of regulatory Tcells, (xii) increases regulatory cell activity and/or one or more ofmyeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromalcells, TIE2-expressing monocytes, (xiii) increases regulatory cellactivity and/or the activity of one or more of myeloid derivedsuppressor cells (MDSCs), iMCs, mesenchymal stromal cells,TIE2-expressing monocytes, (xiii) increases M2 macrophages, (xiv)increases M2 macrophage activity, (xv) increases N2 neutrophils, (xvi)increases N2 neutrophils activity, (xvii) increases inhibition of T cellactivation, (xviii) increases inhibition of CTL activation, (xix)increases inhibition of NK cell activation, (xx) increases T cellexhaustion, (xxi) decreases T cell response, (xxii) decreases activityof cytotoxic cells, (xxiii) reduces antigen-specific memory responses,(xxiv) inhibits apoptosis or lysis of cells, (xxv) decreases cytotoxicor cytostatic effect on cells, (xxvi) reduces direct killing of cells,(xxvii) decreases ThI7 activity, and/or (xxviii) reduces complementdependent cytotoxicity and/or antibody dependent cell-mediatedcytotoxicity, with the proviso that said anti-VISTA antibody orantigen-binding fragment may elicit an opposite effect to one or more of(i)-(xxviii) and optionally is used to treat autoimmunity, allergy,inflammation, transplant or sepsis.

It is a specific object of the invention to provide a method or use ofany agonistic antibody or antibody fragment according to any of theforegoing for use in the treatment or prevention of allergy,autoimmunity, transplant, gene therapy, inflammation, cancer, GVHD orsepsis, or to treat or prevent inflammatory, autoimmune, or allergicside effects associated with any of the foregoing therewith in a humansubject.

An anti-VISTA antibody or antigen-binding fragment or composition, ormethod or use according to any of the foregoing, further comprisinganother immunomodulatory antibody or fusion protein which is selectedfrom immunoinhibitory antibodies or fusion proteins targeting one ormore of CTLA4, PD-1, PDL-1, LAG-3, TIM-3, BTLA, B7-H4, B7-H3, VISTA,and/or agonistic antibodies or fusion protein targeting one or more ofCD40, CD137, OX40, GITR, CD27, CD28 or ICOS.

A method or use of any of the foregoing which includes assaying VISTAprotein by the individual's cells or in bodily fluids prior, concurrentand/or after treatment.

A method or use of any of the foregoing which includes assaying VISTAlevels on hematopoietic cells.

A method or use of any of the foregoing which includes assaying VISTAlevels on hematopoietic cells selected from any one or more of myeloidlineage cells and/or a lymphocytes, monocyte or a neutrophils, T cells,B cells, a natural killer (NK) cells or a natural killer T (NKT) cells.

A method or use of any of the foregoing wherein the agonist anti-humanVISTA antibody or fragment comprises the same CDRs as an antibodyselected from VSTB49-VSTB116 and a human IgG2 Fc region which optionallymay be mutated or wherein the IgG2 constant or Fc region retains nativeFcR binding and/or the ability to bind CD32A.

The antibody, composition, method or use of any of the foregoing whereinthe anti-human VISTA antibody or fragment comprises an affinity or KDfor human VISTA which is 50M or less as determined by surface plasmonresonance at 37° C.

The antibody, composition, method or use of any of the foregoing whereinthe anti-human VISTA antibody or fragment comprises an affinity or KDfor human VISTA which is I nM or less as determined by surface plasmonresonance at 37° c.

It is a specific object of the invention to provide isolatedantagonistic and agonistic anti-human VISTA antibodies and agonisticantibody fragments comprising an antigen binding region thatspecifically binds to human VISTA wherein the antibodies or antibodyfragments comprise variable heavy and light sequences having the CDRpolypeptides of any one of the anti-human VISTA antibodies having thesequences shown in FIG. 4A-4JJ, with the proviso that if said antibodyor fragment comprises an antagonist anti-human VISTA antibody orantibody fragment then the antibody or antibody fragment does notcomprise the same CDRs as any one of VSTB112, VSTB116, VSTB95, VSTB50,VSTB53 or VSTB60.

It is a specific object of the invention to provide isolatedantagonistic and agonistic anti-human VISTA antibodies and agonisticantibody fragments comprising an antigen binding region thatspecifically binds to human VISTA wherein the antibodies or antibodyfragments comprise variable heavy and light sequences having the CDRpolypeptides of an anti-human VISTA antibody selected fromVSTB49-VSTB116, with the proviso that if said antibody or fragmentcomprises an antagonistic anti-human VISTA antibody or anti-human VISTAantibody fragment then the anti-human VISTA antibody or antibodyfragment does not comprise the same CDRs as any one of VSTB112, VSTB116,VSTB95, VSTB50, VSTB53 or VSTB60.

It is another specific object of the invention to provide isolatedantagonistic and agonistic antibodies and antibody fragments comprisingthe CDRs of an anti-human VISTA antibody selected from VSTB49-VSTB116,which comprise a variable heavy and/or variable light polypeptide havingat least 90%, 95%, or 96-99% sequence identity to the variable heavy andlight polypeptide sequences of VSTB49-VSTB116, with the proviso that ifsaid antibody or fragment comprises an antagonistic anti-human VISTAantibody or fragment then the antibody or antibody fragment does notcomprise the same CDRs as any one of VSTB112, VSTB116, VSTB95, VSTB50,VSTB53 or VSTB60.

It is another specific object of the invention to provide isolatedantagonistic and agonistic antibodies or antibody fragments comprisingthe same CDRs any one of VSTB49-VSTB116, which comprise a variable heavyand/or variable light polypeptide which is/are identical to the variableheavy and light polypeptide sequences of VSTB49-VSTB116, with theproviso that if said antibody or fragment comprises an antagonisticanti-human VISTA antibody or fragment then the antibody or antibodyfragment does not comprise the same CDRs as any one of VSTB112, VSTB116,VSTB95, VSTB50, VSTB53 or VSTB60.

It is another specific object of the invention to provide isolatedantagonistic or agonistic chimeric, human, humanized, multispecific(e.g., bispecific) anti-human VISTA antibodies or antibody fragmentscomprising an antigen binding region that specifically binds to humanVISTA which comprise variable heavy and light sequences having the CDRpolypeptides as any one of the anti-human VISTA antibodies comprisingthe CDR and variable heavy and light polypeptides disclosed in FIG.4A-4JJ, with the proviso that if said antibody or fragment comprises anantagonistic anti-human VISTA antibody or antibody fragment then theantibody or antibody fragment does not comprise the same CDRs as any oneof VSTB112, VSTB116, VSTB95, VSTB50, VSTB53 or VSTB60.

It is another specific object of the invention to provide novelimmunosuppressants, i.e., anti-human VISTA antibodies and antibodyfragments, e.g., those containing human IgG2 constant domains or IgG2 Fcregions, optionally wherein the FcR binding capability of the human IgG2constant domains or IgG2 Fc regions are maintained or are enhancedcompared to the wild-type human IgG2 constant domains or IgG2 Fcregions, which agonize, elicit or mimic the effects of human VISTA onimmunity, e.g., its suppressive effects on T cell activity,differentiation and proliferation and its suppressive effects on theexpression of proinflammatory cytokines.

It is another specific object of the invention to provide novelantagonists, i.e., novel anti-human VISTA antibodies and antibodyfragments which antagonize or block the effects of human VISTA onimmunity, particularly its suppressive effects on T cell activity,differentiation and proliferation and its suppressive effects on theexpression of proinflammatory cytokines.

It is another specific object of the invention to provide novelimmunosuppressive antibodies and antibody fragments which enhance ormimic the suppressive effects of VISTA on T cell immunity, i.e., whichsuppress CD4⁺ or CD8⁺ T cell proliferation, CD4⁺ or CD8⁺ T cellactivation and its suppression of the production of immune cytokines,particularly proinflammatory cytokines such as IL-2, IL-4, IL-6, IL-17,TNF-α, and/or GM-CSF (granulocyte-macrophage colony-stimulating factor),and its promoting effects on the expression of chemokines orchemoattractants such as KC (keratinocyte chemoattractant) or MIP-2(Macrophage inflammatory protein 2).

It is another specific object of the invention to provide novelantibodies and antibody fragments which block or reduce the suppressiveeffects of VISTA on T cell immunity, i.e., which enhance CD4⁺ or CD8⁺ Tcell proliferation, CD4⁺ or CD8⁺ T cell activation, and its suppressiveeffects on the production of proinflammatory immune cytokines,particularly proinflammatory cytokines such as IL-2, IL-4, IL-6, IL-17,TNF-a, and/or GM-CSF (granulocyte-macrophage colony-stimulating factor),and its promoting effects on the expression of chemokines orchemoattractants such as KC (keratinocyte chemoattractant) or MIP-2(Macrophage inflammatory protein 2).

It is another specific object of the invention to provide novelimmunosuppressive or agonistic anti-human VISTA antibodies and antibodyfragments of specific epitopic specificity or which compete for bindingto human VISTA with specific anti-human VISTA antibodies.

It is another specific object of the invention to provide novelimmunosuppressive or agonistic anti-human VISTA antibodies and antibodyfragments of specific epitopic specificity or which compete for bindingto human VISTA with specific anti-human VISTA antibodies which agonize(enhance, elicit or mimic) the suppressive effects of VISTA on immunity,e.g., its suppressive effects on T cell immunity, i.e., CD4⁺ or CD8+ Tcell proliferation, CD4⁺ or CD8⁺ T cell activation, and/or whichsuppress the production of proinflammatory immune cytokines such asIL-2, IL-4, IL-6, IL-17, TNF-a, and/or GM-CSF (granulocyte-macrophagecolony-stimulating factor), and its promoting effects on the expressionof chemokines or chemoattractants such as KC (keratinocytechemoattractant) or MIP-2 (Macrophage inflammatory protein 2).

Also the invention also relates to the specific use of these agonisticanti-human VISTA antibodies and antibody fragments as prophylactics ortherapeutics, especially in treating conditions wherein preventing orinhibiting or reducing immune reactions is therapeutically desirable,and more particularly wherein the preventing or inhibiting or reducing Tcell immunity, or more specifically CD4⁺ or CD8⁺ mediated T cellimmunity is therapeutically beneficial such as autoimmunity,inflammation, allergic disorders, sepsis, GVHD, and/or in treatingtransplant or cell therapy recipients, e.g., CAR-T recipients, or inalleviating the inflammatory side effects of some conditions such ascancer.

Also the invention relates to the use of novel antagonistic anti-humanVISTA antibodies and antibody fragments as prophylactics ortherapeutics, especially in treating conditions wherein promotingimmunity is desired, e.g., T cell immunity or CD4⁺ or CD8⁺-mediated Tcell immunity is therapeutically beneficial such as cancer andinfectious disease.

It is another specific object of the invention to provide an agonist orantagonist anti-human VISTA antibody according to the invention which isattached to a detectable label, linker or a therapeutic moiety.

It is another specific object of the invention to provide a diagnosticor therapeutic composition comprising a diagnostically ortherapeutically effective amount of an agonist or antagonist anti-humanVISTA antibody according to the invention, e.g., one containing the sameCDRs as any of the antibodies having the sequences shown in FIG. 4A-4JJwhich is suitable for use in human therapy, such as an intravenous,subcutaneous or intramuscular administrable composition.

It is another specific object of the invention to provide a diagnosticor therapeutic methods which use an agonist antibody according to theinvention in association with another immune agonist, e.g., a PD-1 orPD-LI agonist, e.g., wherein the PD-1 or PD-LI agonist is selected froman anti-PD-1 antibody or antibody fragment, an anti-PD-L1 antibody orantibody fragment, a PD-LI polypeptide or fragment thereof which may bemonovalent or multimeric, a PD-1 polypeptide or fragment thereof whichmay be monovalent or multimeric, or a complex or fusion proteincomprising any of the foregoing.

It is another specific object of the invention to provide diagnostic ortherapeutic methods which use an antagonist antibody according to theinvention in association with another immune antagonist, e.g., a PD-1 orPD-LI antagonist, e.g., wherein the PD-1 or PD-LI agonist is selectedfrom an antagonist anti-PD-1 antibody or antibody fragment, anantagonist anti-PD-LI antibody or antibody fragment.

It is another specific object of the invention to provide methods ofcontacting immune cells in vitro or in vivo with an antagonist oragonist antibody according to the invention, e.g., human immune cells,e.g., wherein the contacted cells are infused into a human subject suchas a subject who has cancer or an infectious disease or one who has aninflammatory, allergic or autoimmune condition.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-D. This figure shows in vitro and in vivo screening assays whichcan be used to identify suppressive VISTA mAbs. A) Purified T cells wereplated on top of anti-CD3 in the presence of the indicated mAb for 72hours. Proliferation was measured by H3 incorporation. B) PurifiedDO11.10 T cells were stimulated by ISQ pulsed APCs for 6 days in thepresence of the indicated antibody. Proliferation was measured throughuse of CTV dilution dye. C) GVHD was induced by transfer of C57BL/6cells into irradiated BALB/c recipients. Mice were injected I.P. with200 μg of antibody on day 0, 2 and 4 post transfer and survival wasanalyzed. D) Mice were treated with 10 mpk of the indicated antibody 3hours prior to administration of ConA (15 mpk) and IL-2 was analyzed inplasma at 6 by Luminex.

FIG. 2A-F. This figure shows that agonist VISTA antibodies areimmunosuppressive in multiple models of autoimmune disease. A) NZB/W FImice were treated 3×/week with either 8G8 or Ham Ig (200 μg) starting at25 weeks until the end of the experiment. “X” denotes time points wherethe control treated group had all been sacrificed. B) Mice were treatedwith 200 μg of antibody 3 hours prior to administration of 15 mg/kg(mpk) of ConA and survival was followed for 80 hours. C) Mice weretreated sequentially with Collagen II mAb followed by LPS and arthritiswas measured by measuring for paw swelling. 8G8 and Ham-Ig wereadministered (200 μg) 3× every other day. D) Imiquimod was applied tothe ear of mice daily. At day 14, 8G8 or Ham-Ig (200 μg) wereadministered every other day and ear thickness was measured withcalipers. E, F) Imiquimod was applied to the backs of mice daily. At day9, mice were euthanized and skin was sectioned & stained for CD3expression by IHC.

FIG. 3 . This figure shows the expression of VISTA in WT and hV-KI mice.CD4+ T cells, CD8⁺ T cells, Tregs (CD4⁺ FoxP3⁺), and monocytes, CDIIb⁺,Ly6C⁺, Ly6G⁻ were isolated from the lymph nodes of WT and VISTA KI mice,and stained with a VISTA antibodies against mouse or human proteinrespectively.

FIG. 4A-4JJ contains the sequences of different anti-human VISTAantibodies including those of INX800, INX801, and INX900-INX919.

FIG. 5 shows the effects of exemplary anti-human VISTA antibodies, i.e.,INX800 and INX801 in a ConA hepatitis model which assesses the effectsthereof on the expression of different cytokines, chemokines andchemoattractants.

FIG. 6 shows the effects of exemplary anti-human VISTA antibodies, i.e.,INX800 and INX801 in an in vivo graft versus host disease (GVHD) animalmodel.

FIG. 7 shows the effects of exemplary agonistic anti-human VISTAantibodies, i.e., INX800 or INX801 on CD3-driven T cell immuneresponses.

FIG. 8 shows the effects of exemplary agonistic anti-human VISTAantibodies, i.e., INX800 or INX801 on the number of specific T cellpopulations or on total T cell numbers.

FIG. 9 compares the effects of exemplary anti-human VISTA antibodies inConA assays and on the expression of select proinflammatory cytokinesand inflammation markers, i.e., IL-2, γ interferon and IL-12p70.

FIG. 10A-C: shows different IgG2 Isoforms. (A) Disulfide shuffling leadsto isoforms A and B, along with the transition for A/B (figure fromZhang, A. et al., 2015). (B) Isoforms are distinguishable by RP-HPLC.(C) Observed RP-HPLC chromatogram for INX901.

FIG. 11 : shows chemical enrichment of IgG2 A or B isoforms. (Blackline, top) Chromatogram shows a dominant left-most peak defining theB-form. (Red line, bottom) Chromatogram shows a dominant right peakdefining the A-form.

FIG. 12 : compares INX901 Fc-silent variants with respect to disulfideshuffling. (Top) INX901 on an IgG2 backbone exhibits an expected mixtureof A, A/B, and B isoforms. (Middle) INX901Si on a silent IgGI backboneexists as a single isoform. (Bottom) INX901HSi possesses an IgGI silentFc region with a CHI/hinge from IgG2, which enables disulfide shufflingequivalent to native IgG2.

FIG. 13 . Biochemically skewed INX901 forms can still reduce cytokineproduction in the MLR. Supernatants from two separate MLRs were analyzedfor cytokine production at the 72-hour time point by Luminex analysis.INX901 parental, A skew and B skew all reduced the production of TNFaand IL-2 in a dose dependent fashion.

FIG. 14 . Genetically locked INX901 forms can still reduce cytokineproduction in the MLR, but Fc silent variants cannot. Supernatants fromeach MLR were analyzed for cytokine production at the 72-hour time pointby Luminex analysis. INX901 parental, A lock and B lock all reduced theproduction of TNFa and IL-2 in a dose dependent fashion. The Si and HSivariants, which contain mutations to silence the Fc domain, did notconsistently suppress cytokine production.

FIG. 15 . Genetically locked INX908 forms can still reduce cytokineproduction in the MLR, but Fc silent variants cannot. Supernatants fromeach MLR were analyzed for cytokine production at the 72-hour time pointby Luminex analysis. INX908 parental, A lock and B lock all reduced theproduction of TNFa and IL-2 in a dose dependent fashion. The si and HSivariants, which contain mutations to silence the Fc domain, did notconsistently suppress cytokine production.

FIG. 16 . This figure schematically describes the Pepscan® technologyused to identify linear and discontinuous epitopes bound by agonistanti-human VISTA antibodies.

FIG. 17 : This figure shows that agonist anti-human VISTA antibodiesbind to the same core sequence.

FIG. 18 : This figure summarizes the epitope analysis for differentanti-human VISTA antibodies according to the invention.

FIG. 19 : This figure shows the epitopes bound by agonist anti-humanVISTA antibodies and further identifies important residues involved inbinding.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein may be used inthe invention or testing of the present invention, suitable methods andmaterials are described herein. The materials, methods and examples areillustrative only, and are not intended to be limiting. Thenomenclatures utilized in connection with, and the laboratory proceduresand techniques of, analytical chemistry, synthetic organic chemistry,and medicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques may be usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise.

“Activating receptor,” as used herein, refers broadly to immune cellreceptors that bind antigen, complexed antigen (e.g., in the context ofMHC molecules), Ig-fusion proteins, ligands, or antibodies. Activatingreceptors but are not limited to T cell receptors (TCRs), B cellreceptors (BCRs), cytokine receptors, LPS receptors, complementreceptors, and Fc receptors. For example, T cell receptors are presenton T cells and are associated with CD3 molecules. T cell receptors arestimulated by antigen in the context of MHC molecules (as well as bypolyclonal T cell activating reagents). T cell activation via the TCRresults in numerous changes, e.g., protein phosphorylation, membranelipid changes, ion fluxes, cyclic nucleotide alterations, RNAtranscription changes, protein synthesis changes, and cell volumechanges. For example, T cell receptors are present on T cells and areassociated with CD3 molecules. T cell receptors are stimulated byantigen in the context of MHC molecules (as well as by polyclonal T cellactivating reagents). T cell activation via the TCR results in numerouschanges, e.g., protein phosphorylation, membrane lipid changes, ionfluxes, cyclic nucleotide alterations, RNA transcription changes,protein synthesis changes, and cell volume changes.

“Adjuvant” as used herein, refers to an agent used to stimulate theimmune system and increase the response to a vaccine, without having anyspecific antigenic effect in itself.

“Agonist” herein refers to a molecule, generally an antibody or fusionproteins which enhances or mimics the effects of a specific molecule onimmunity. Generally in the present application this will refer toanti-human VISTA agonist antibodies and antibody fragments which enhanceor mimic the effects of human VISTA on immunity, particularly VISTA'ssuppressive effects on T cell immunity (CD4+ and/or CD8+ T cellimmunity), the expression of proinflammatory cytokines and its effectsof the expression of specific chemokines and chemoattractants.

“Aids in the diagnosis” or “aids in the detection” of a disease hereinmeans that the expression level of a particular marker polypeptide orexpressed RNA is detected alone or in association with one or more othermarkers in order to assess whether a subject has cells characteristic ofa particular disease condition or the onset of a particular diseasecondition or comprises immune dysfunction such as immunosuppressioncharacterized by VISTA expression or abnormal immune upregulationcharacterized by cells having reduced VISTA levels, such as duringautoimmunity, inflammation or allergic responses, e.g., in individualswith chronic and non-chronic diseases.

“Allergic disease,” as used herein, refers broadly to a diseaseinvolving allergic reactions. More specifically, an “allergic disease”is defined as a disease for which an allergen is identified, where thereis a strong correlation between exposure to that allergen and the onsetof pathological change, and where that pathological change has beenproven to have an immunological mechanism. Herein, an immunologicalmechanism means that leukocytes show an immune response to allergenstimulation.

“Amino acid,” as used herein refers broadly to naturally occurring andsynthetic amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those amino acids that are later modified(e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.) Aminoacid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid (i.e., a carbon that isbound to a hydrogen, a carboxyl group, an amino group), and an R group(e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium.) Analogs may have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

“Anergy” or “tolerance,” or “prolonged antigen-specific T cellsuppression” or “prolonged immunosuppression” as used herein refersbroadly to refractivity to activating receptor-mediated stimulation.Refractivity is generally antigen-specific and persists after exposureto the tolerizing antigen has ceased. For example, anergy in T cells (asopposed to unresponsiveness) is characterized by lack of cytokineproduction, e.g., IL-2. T cell anergy occurs when T cells are exposed toantigen and receive a first signal (a T cell receptor or CD-3 mediatedsignal) in the absence of a second signal (a costimulatory signal).Under these conditions, reexposure of the cells to the same antigen(even if reexposure occurs in the presence of a costimulatory molecule)results in failure to produce cytokines and, thus, failure toproliferate. Anergic T cells can, however, mount responses to unrelatedantigens and can proliferate if cultured with cytokines (e.g., IL-2).For example, T cell anergy can also be observed by the lack of IL-2production by T lymphocytes as measured by ELISA or by a proliferationassay using an indicator cell line. Alternatively, a reporter geneconstruct can be used. For example, anergic T cells fail to initiateIL-2 gene transcription induced by a heterologous promoter under thecontrol of the 5′ IL-2 gene enhancer or by a multimer of the APIsequence that can be found within the enhancer (Kang et al. (1992)Science 257: 1134). Modulation of a costimulatory signal results inmodulation of effector function of an immune cell.

“Antagonist” herein refers to a molecule, generally an antibody orfusion proteins which blocks or reduces the effects of a specificmolecule on immunity. Generally in the present application this willrefer to anti-human VISTA antagonist antibodies and antibody fragmentswhich block or reduce the effects of human VISTA on immunity,particularly VISTA's suppressive effects on T cell immunity (CD4⁺ and/orCD8+ T cell immunity), the expression of proinflammatory cytokines andVISTA's effects of the expression of specific chemokines andchemoattractants.

“Antibody”, as used herein, refers broadly to an “antigen-bindingportion” of an antibody (also used interchangeably with “antibodyportion,” “antigen-binding fragment,” “antibody fragment”), as well aswhole antibody molecules. The term “antigen-binding portion”, as usedherein, refers to one or more fragments of an antibody that retain theability to specifically bind to an antigen (e.g., VISTA or specificportions thereof)). The term “antibody” as referred to herein includeswhole polyclonal and monoclonal antibodies and any antigen-bindingfragment (i.e., “antigen-binding portion”) or single chains thereof aswell as bispecific and multispecific antibodies, e.g., those that bindto multiple antigens or multiple antigen epitopes. An “antibody” refersto a glycoprotein comprising at least two heavy (H) chains and two light(L) chains interconnected by disulfide bonds, or an antigen-bindingportion thereof. Each heavy chain is comprised of at least one heavychain variable region (abbreviated herein as V_(H)) and a heavy chainconstant region. The heavy chain constant region is comprised of threedomains, C_(H)i, Cm and Cm- Each light chain is comprised of at leastone light chain variable region (abbreviated herein as V_(L)) and alight chain constant region. The light chain constant region iscomprised of one domain, CL—The V_(H) and V_(L) regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FRs). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. The variable regions of the heavy and light chainscontain a binding domain that interacts with an antigen. The constantregions of the antibodies may mediate the binding of the immunoglobulinto host tissues or factors, including various cells of the immune system(e.g., effector cells) and the first component (Clq) of the classicalcomplement system. More generally, the term “antibody” is intended toinclude any polypeptide chain-containing molecular structure with aspecific shape that fits to and recognizes an epitope, where one or morenon-covalent binding interactions stabilize the complex between themolecular structure and the epitope. The archetypal antibody molecule isthe immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA,IgE, IgD, etc., from all sources, e.g. human, rodent, rabbit, cow,sheep, pig, dog, other mammals, chicken, other avians, etc., areconsidered to be “antibodies.”

The antigen-binding function of an antibody can be performed byfragments of a full-length antibody. Non-limiting examples ofantigen-binding fragments encompassed within the term “antigen-bindingportion” of an antibody include (a) a Fab fragment, a monovalentfragment consisting of the V_(L), V_(H), C_(L) and C_(Hi) domains; (b) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (c) a Fd fragmentconsisting of the V_(H) and C_(Hi) domains; (d) a Fv fragment consistingof the VL and VH domains of a single arm of an antibody; (e) a dAbfragment (Ward, et al. (1989) Nature 341:544-546), which consists of aV_(H) domain; and (f) an isolated complementarily determining region(CDR). Furthermore, although the two domains of the Fv fragment, V_(L)and V_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv). See e.g.,Bird, et al. (1988) Science 242: 423-426; Huston, et al. (1988) ProcNatl. Acad. Sci. USA 85: 5879-5883; and Osbourn, et al. (1998) Nat.Biotechnol. 16: 778. Single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Any V_(H) and V_(L) sequences of specific scFv can be linked to humanimmunoglobulin constant region cDNA or genomic sequences, in order togenerate expression vectors encoding complete IgG molecules or otherisotypes. VH and V_(L) can also be used in the generation of Fab, Fv, orother fragments of immunoglobulins using either protein chemistry orrecombinant DNA technology. Other forms of single chain antibodies, suchas diabodies are also encompassed. Diabodies are bivalent, bispecificantibodies in which V_(H) and V_(L) domains are expressed on a singlepolypeptide chain, but using a linker that is too short to allow forpairing between the two domains on the same chain, thereby forcing thedomains to pair with complementary domains of another chain and creatingtwo antigen-binding sites. See e.g. Holliger, et al. (1993) Proc Natl.Acad. Sci. USA 90: 6444-6448; Poljak, et al. (1994) Structure2:1121-1123. Still further, an antibody or antigen-binding portionthereof (antigen-binding fragment, antibody fragment, antibody portion)may be part of a larger immunoadhesion molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of immunoadhesion moleculesinclude use of the streptavidin core region to make a tetrameric scFvmolecule (Kipriyanov, et al. (1995) Hum. Antibodies Hybridomas 6:93-101)and use of a cysteine residue, a marker peptide and a C-terminalpolyhistidine tag to make bivalent and biotinylated scFv molecules.Kipriyanov, et al. (1994) Mol. Immunol. 31: 1047-1058. Antibodyportions, such as Fab and F(ab′)₂ fragments, can be prepared from wholeantibodies using conventional techniques, such as papain or pepsindigestion, respectively, of whole antibodies. Moreover, antibodies,antibody portions and immunoadhesion molecules can be obtained usingstandard recombinant DNA techniques, as described herein. Antibodies maybe polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, ormodified forms thereof, e.g., humanized, chimeric, bispecific ormultispecific antibodies.

“Antibody recognizing an antigen” and “an antibody specific for anantigen” is used interchangeably herein with the term “an antibody whichbinds specifically to an antigen” and refers to an immunoglobulin orfragment thereof that specifically binds an antigen.

“Antigen,” as used herein, refers broadly to a molecule or a portion ofa molecule capable of being bound by an antibody which is additionallycapable of inducing an animal to produce an antibody capable of bindingto an epitope of that antigen. An antigen may have one epitope, or havemore than one epitope. The specific reaction referred to hereinindicates that the antigen will react, in a highly selective manner,with its corresponding antibody and not with the multitude of otherantibodies which may be evoked by other antigens. In the case of adesired enhanced immune response to particular antigens of interest,antigens include, but are not limited to; infectious disease antigensfor which a protective immune response may be elicited are exemplary.

“Antigen presenting cell,” as used herein, refers broadly toprofessional antigen presenting cells (e.g., B lymphocytes, monocytes,dendritic cells, and Langerhans cells) as well as other antigenpresenting cells (e.g., keratinocytes, endothelial cells, astrocytes,fibroblasts, and oligodendrocytes).

“Antisense nucleic acid molecule,” as used herein, refers broadly to anucleotide sequence which is complementary to a “sense” nucleic acidencoding a protein (e.g., complementary to the coding strand of adouble-stranded cDNA molecule) complementary to an m RNA sequence orcomplementary to the coding strand of a gene. Accordingly, antisensenucleic acid molecules can hydrogen bond to sense nucleic acidmolecules.

“Apoptosis,” as used herein, refers broadly to programmed cell deathwhich can be characterized using techniques which are known in the art.Apoptotic cell death can be characterized by cell shrinkage, membraneblebbing, and chromatin condensation culminating in cell fragmentation.Cells undergoing apoptosis also display a characteristic pattern ofinternucleosomal DNA cleavage.

“Autoimmunity” or “autoimmune disease or condition,” as used herein,refers broadly to a disease or disorder arising from and directedagainst an individual's own tissues or a co-segregate or manifestationthereof or resulting condition therefrom, and includes. Hereinautoimmune conditions include inflammatory or allergic conditions, e.g.,chronic diseases characterized by a host immune reaction againstself-antigens potentially associated with tissue destruction such asrheumatoid arthritis.

“B cell receptor” (BCR),” as used herein, refers broadly to the complexbetween membrane Ig (mIg) and other transmembrane polypeptides (e.g.,IgA. and Ig) found on B cells. The signal transduction function of mIgis triggered by crosslinking of receptor molecules by oligomeric ormultimeric antigens. B cells can also be activated byanti-immunoglobulin antibodies. Upon BCR activation, numerous changesoccur in B cells, including tyrosine phosphorylation.

“Cancer,” as used herein, refers broadly to any neoplastic disease(whether invasive or metastatic) characterized by abnormal anduncontrolled cell division causing malignant growth or tumor (e.g.,unregulated cell growth.) The term “cancer” or “cancerous” as usedherein should be understood to encompass any neoplastic disease (whetherinvasive, non-invasive or metastatic) which is characterized by abnormaland uncontrolled cell division causing malignant growth or tumor,non-limiting examples of which are described herein. This includes anyphysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer are exemplified in theworking examples. Further cancers include but are not limited to,carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particularexamples of such cancers include squamous cell cancer, lung cancer(including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung), cancerof the peritoneum, hepatocellular cancer, gastric or stomach cancer(including gastrointestinal cancer), pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastsleukemia; multiple myeloma and post-transplant lymphoproliferativedisorder (PTLD). Other cancers amenable for treatment by the presentinvention include, but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include colorectal, bladder,ovarian, melanoma, squamous cell cancer, lung cancer (includingsmall-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, and squamous carcinoma of the lung), cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer (includinggastrointestinal cancer), pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.Preferably, the cancer is selected from the group consisting ofcolorectal cancer, breast cancer, colorectal cancer, rectal cancer,non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), renal cellcancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissuesarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer,melanoma, ovarian cancer, mesothelioma, and multiple myeloma. In anexemplary embodiment the cancer is an early or advanced (includingmetastatic) bladder, ovarian or melanoma. In another embodiment thecancer is colorectal cancer. The cancerous conditions amenable fortreatment of the invention include cancers that express or do notexpress VISTA and further include non-metastatic or non-invasive as wellas invasive or metastatic cancers wherein VISTA expression by immune,stromal or diseased cells suppress antitumor responses and anti-invasiveimmune responses. The method of the present invention is particularlysuitable for the treatment of vascularized tumors. Cancers according tothe invention include cancers that express or do not express VISTA andfurther include non-metastatic or non-invasive as well as invasive ormetastatic cancers wherein VISTA expression by immune, stromal ordiseased cells suppress antitumor responses and anti-invasive immuneresponses, and those characterized by vascularized tumors.

“Chimeric antibody,” as used herein, refers broadly to an antibodymolecule in which the constant region, or a portion thereof, is altered,replaced or exchanged so that the antigen-binding site (variable region)is linked to a constant region of a different or altered class, effectorfunction and/or species, or an entirely different molecule which confersnew properties to the chimeric antibody, e.g., an enzyme, toxin,hormone, growth factor, drug, the variable region or a portion thereof,is altered, replaced or exchanged with a variable region having adifferent or altered antigen specificity.

“Coding region,” as used herein, refers broadly to regions of anucleotide sequence comprising codons which are translated into aminoacid residues, whereas the term “noncoding region” refers to regions ofa nucleotide sequence that are not translated into amino acids (e.g., 5′and 3′ untranslated regions).

“Conservatively modified variants,” as used herein, applies to bothamino acid and nucleic acid sequences, and with respect to particularnucleic acid sequences, refers broadly to conservatively modifiedvariants refers to those nucleic acids which encode identical oressentially identical amino acid sequences, or where the nucleic aciddoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given protein.“Silent variations” are one species of conservatively modified nucleicacid variations. Every nucleic acid sequence herein which encodes apolypeptide also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) may be modifiedto yield a functionally identical molecule.

“Complementarity determining region,” “hypervariable region,” or “CDR,”as used herein, refers broadly to one or more of the hyper-variable orcomplementarily determining regions (CDRs) found in the variable regionsof light or heavy chains of an antibody. See Kabat, et al. (1987)Sequences of Proteins of Immunological Interest National Institutes ofHealth, Bethesda, Md. These expressions include the hypervariableregions as defined by Kabat, et al. (1983) Sequences of Proteins ofImmunological Interest, U.S. Dept. of Health and Human Services or thehypervariable loops in 3-dimensional structures of antibodies. Chothiaand Lesk (1987) J. Mol. Biol. 196: 901-917. The CDRs in each chain areheld in close proximity by framework regions and, with the CDRs from theother chain, contribute to the formation of the antigen-binding site.Within the CDRs there are select amino acids that have been described asthe selectivity determining regions (SDRs) which represent the criticalcontact residues used by the CDR in the antibody-antigen interaction.(Kashmiri Methods 36: 25-34 (2005)).

“Control amount,” as used herein, refers broadly to a marker can be anyamount or a range of amounts to be compared against a test amount of amarker. For example, a control amount of a marker may be the amount of amarker in a patient with a particular disease or condition or a personwithout such a disease or condition. A control amount can be either inabsolute amount (e.g., microgram/ml) or a relative amount (e.g.,relative intensity of signals).

“Costimulatory receptor,” as used herein, refers broadly to receptorswhich transmit a costimulatory signal to an immune cell, e.g., CD28 orICOS. As used herein, the term “inhibitory receptors” includes receptorswhich transmit a negative signal to an immune cell, e.g., a T cell or anNK cell.

“Costimulate,” as used herein, refers broadly to the ability of acostimulatory molecule to provide a second, non-activating,receptor-mediated signal (a “costimulatory signal”) that inducesproliferation or effector function. For example, a costimulatory signalcan result in cytokine secretion (e.g., in a T cell that has received aT cell-receptor-mediated signal) Immune cells that have received a cellreceptor-mediated signal (e.g., via an activating receptor) may bereferred to herein as “activated immune cells.” With respect to T cells,transmission of a costimulatory signal to a T cell involves a signalingpathway that is not inhibited by cyclosporin A. In addition, acostimulatory signal can induce cytokine secretion (e.g., IL-2 and/orIL-10) in a T cell and/or can prevent the induction of unresponsivenessto antigen, the induction of anergy, or the induction of cell death inthe T cell.

“Costimulatory polypeptide” or “costimulatory molecule” herein refers toa polypeptide that, upon interaction with a cell-surface molecule on Tcells, modulates T cell responses.

“Costimulatory signaling” as used herein is the signaling activityresulting from the interaction between costimulatory polypeptides onantigen presenting cells and their receptors on T cells duringantigen-specific T cell responses. Without wishing to be limited by asingle hypothesis, the antigen-specific T cell response is believed tobe mediated by two signals: 1) engagement of the T cell Receptor (TCR)with antigenic peptide presented in the context of MHC (signal 1), and2) a second antigen-independent signal delivered by contact betweendifferent costimulatory receptor/ligand pairs (signal 2). Withoutwishing to be limited by a single hypothesis, this “second signal” iscritical in determining the type of T cell response (activation vsinhibition) as well as the strength and duration of that response, andis regulated by both positive and negative signals from costimulatorymolecules, such as the B7 family of proteins.

“B7” polypeptide herein means a member of the B7 family of proteins thatcostimulate T cells including, but not limited to B7-1, B7-2, B7-DC,B7-H5, B7-HI, B7-H2, B7-H3, B7-H4, B7-H6, B7-S3 and biologically activefragments and/or variants thereof. Representative biologically activefragments include the extracellular domain or fragments of theextracellular domain that costimulate T cells.

“Cytoplasmic domain,” as used herein, refers broadly to the portion of aprotein which extends into the cytoplasm of a cell.

“Diagnostic,” as used herein, refers broadly to identifying the presenceor nature of a pathologic condition. Diagnostic methods differ in theirsensitivity and specificity. The “sensitivity” of a diagnostic assay isthe percentage of diseased individuals who test positive (percent of“true positives”). Diseased individuals not detected by the assay are“false negatives.” Subjects who are not diseased and who test negativein the assay are termed “true negatives.” The “specificity” of adiagnostic assay is 1 minus the false positive rate, where the “falsepositive” rate is defined as the proportion of those without the diseasewho test positive. While a particular diagnostic method may not providea definitive diagnosis of a condition, it suffices if the methodprovides a positive indication that aids in diagnosis.

“Diagnosing,” or “aiding in the diagnosis” as used herein refers broadlyto classifying a disease or a symptom, and/or determining the likelihoodthat an individual has a disease condition (e.g., based on absence orpresence of VISTA expression, and/or increased or decreased expressionby immune, stromal and/or putative diseased cells); determining aseverity of the disease, monitoring disease progression, forecasting anoutcome of a disease and/or prospects of recovery. The term “detecting”may also optionally encompass any of the foregoing. Diagnosis of adisease according to the present invention may, in some embodiments, beaffected by determining a level of a polynucleotide or a polypeptide ofthe present invention in a biological sample obtained from the subject,wherein the level determined can be correlated with predisposition to,or presence or absence of the disease. It should be noted that a“biological sample obtained from the subject” may also optionallycomprise a sample that has not been physically removed from the subject.

“Effective amount,” as used herein, refers broadly to the amount of acompound, antibody, antigen, or cells that, when administered to apatient for treating a disease, is sufficient to effect such treatmentfor the disease. The effective amount may be an amount effective forprophylaxis, and/or an amount effective for prevention. The effectiveamount may be an amount effective to reduce, an amount effective toprevent the incidence of signs/symptoms, to reduce the severity of theincidence of signs/symptoms, to eliminate the incidence ofsigns/symptoms, to slow the development of the incidence ofsigns/symptoms, to prevent the development of the incidence ofsigns/symptoms, and/or effect prophylaxis of the incidence ofsigns/symptoms. The “effective amount” may vary depending on the diseaseand its severity and the age, weight, medical history, susceptibility,and pre-existing conditions, of the patient to be treated. The term“effective amount” is synonymous with “therapeutically effective amount”for purposes of this invention.

“Extracellular domain” or “ECD” as used herein refers broadly to theportion of a protein that extends from the surface of a cell.

“Expression vector,” as used herein, refers broadly to any recombinantexpression system for the purpose of expressing a nucleic acid sequenceof the invention in vitro or in vivo, constitutively or inducibly, inany cell, including prokaryotic, yeast, fungal, plant, insect ormammalian cell. The term includes linear or circular expression systems.The term includes expression systems that remain episomal or integrateinto the host cell genome. The expression systems can have the abilityto self-replicate or not, i.e., drive only transient expression in acell. The term includes recombinant expression cassettes which containonly the minimum elements needed for transcription of the recombinantnucleic acid.

“Family,” as used herein, refers broadly to the polypeptide and nucleicacid molecules of the invention is intended to mean two or morepolypeptide or nucleic acid molecules having a common structural domainor motif and having sufficient amino acid or nucleotide sequencehomology as defined herein. Family members can be naturally ornon-naturally occurring and can be from either the same or differentspecies. For example, a family can contain a first polypeptide of humanorigin, as well as other, distinct polypeptides of human origin oralternatively, can contain homologues of non-human origin (e.g., monkeypolypeptides.) Members of a family may also have common functionalcharacteristics.

“Fc receptor” (FcRs) as used herein, refers broadly to cell surfacereceptors for the Fc portion of immunoglobulin molecules (Igs). Fcreceptors are found on many cells which participate in immune responses.Among the human FcRs that have been identified so far are those whichrecognize IgG (designated FcyR), IgE (FceRI), IgA (FcaR), andpolymerized IgM/A (FcεμP). FcRs are found in the following cell types:FceRI (mast cells), FceRII (many leukocytes), FcaR (neutrophils), andFcμR (glandular epithelium, hepatocytes). (Hogg Immunol. Today 9: 185-86(1988)). The widely studied FcyRs are central in cellular immunedefenses, and are responsible for stimulating the release of mediatorsof inflammation and hydrolytic enzymes involved in the pathogenesis ofautoimmune disease. (Unkeless, Annu. Rev. Immunol. 6:251-87 (1988)). TheFcyRs provide a crucial link between effector cells and the lymphocytesthat secrete Ig, since the macrophage/monocyte, polymorphonuclearleukocyte, and natural killer (NK) cell FcyRs confer an element ofspecific recognition mediated by IgG. Human leukocytes have at leastthree different types of FcyRs for IgG: hFcyRI (CD64) (found onmonocytes/macrophages), hFcyRIIA or hFcyRIIB, (CD32 or CD32A) (found onmonocytes, neutrophils, eosinophils, platelets, possibly B cells, andthe K562 cell line) and FcyRIIIA (CD16A) or FcyRIIIB (CD16B) (found onNK cells, neutrophils, eosinophils, and macrophages).

“Framework region” or “FR,” as used herein refers broadly to one or moreof the framework regions within the variable regions of the light andheavy chains of an antibody. See Kabat, et al. Sequences of Proteins ofImmunological Interest National Institutes of Health, Bethesda, Md.(1987). These expressions include those amino acid sequence regionsinterposed between the CDRs within the variable regions of the light andheavy chains of an antibody.

“Heterologous,” as used herein, refers broadly to portions of a nucleicacid indicates that the nucleic acid comprises two or more subsequencesthat are not found in the same relationship to each other in nature. Forinstance, the nucleic acid is typically recombinantly produced, havingtwo or more sequences from unrelated genes arranged to make a newfunctional nucleic acid (e.g., a promoter from one source and a codingregion from another source.) Similarly, a heterologous protein indicatesthat the protein comprises two or more subsequences that are not foundin the same relationship to each other in nature (e.g., a fusionprotein).

“High affinity,” as used herein, refers broadly to an antibody or fusionprotein having a KD of at least 10⁻⁵ M, more preferably 10⁻⁷ M, evenmore preferably at least 10⁻⁸ M and even more preferably at least 10⁻⁹M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M for a target antigen or receptor. “Highaffinity” for an IgG antibody or fusion protein herein refers to anantibody having a KD of 10⁻⁶ M or less, more preferably 10⁻⁷ M or less,preferably 10⁻⁸ M or less, more preferably 10⁻⁹ M or less and even morepreferably 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M or less for a target antigen orreceptor. With particular respect to antibodies, “high affinity” bindingcan vary for different antibody isotypes. For example, “high affinity”binding for an IgM isotype refers to an antibody having a K_(D) of 10⁻⁷M or less, more preferably 10⁻⁸ M or less.

“Homology,” as used herein, refers broadly to a degree of similaritybetween a nucleic acid sequence and a reference nucleic acid sequence orbetween a polypeptide sequence and a reference polypeptide sequence.Homology may be partial or complete. Complete homology indicates thatthe nucleic acid or amino acid sequences are identical. A partiallyhomologous nucleic acid or amino acid sequence is one that is notidentical to the reference nucleic acid or amino acid sequence. Thedegree of homology can be determined by sequence comparison, for exampleusing BlastP software of the National Center of BiotechnologyInformation (NCBI) using default parameters. The term “sequenceidentity” may be used interchangeably with “homology.”

“Host cell,” as used herein, refers broadly to refer to a cell intowhich a nucleic acid molecule of the invention, such as a recombinantexpression vector of the invention, has been introduced. Host cells maybe prokaryotic cells (e.g., E. coli), or eukaryotic cells such as yeast,insect (e.g., SF9), amphibian, or mammalian cells such as CHO, HeLa,HEK-293, e.g., cultured cells, explants, and cells in vivo. The terms“host cell” and “recombinant host cell” are used interchangeably herein.It should be understood that such terms refer not only to the particularsubject cell but to the progeny or potential progeny of such a cell.Because certain modifications may occur in succeeding generations due toeither mutation or environmental influences, progeny may not, in fact,be identical to the parent cell, but are still included within the scopeof the term as used herein.

“Human monoclonal antibody” refers to antibodies displaying a singlebinding specificity which have variable regions in which both theframework and CDR regions are derived from human germ lineimmunoglobulin sequences. In one embodiment, the human monoclonalantibodies are produced by a hybridoma which includes a B cell obtainedfrom a transgenic non human animal, e.g., a transgenic mouse, having agenome comprising a human heavy chain transgene and a light chaintransgene fused to an immortalized cell. This includes fully humanmonoclonal antibodies and conjugates and variants thereof, e.g., whichare bound to effector agents such as therapeutics or diagnostic agents.

“Humanized antibody,” as used herein, refers broadly to includeantibodies made by a non-human cell having variable and constant regionswhich have been altered to more closely resemble antibodies that wouldbe made by a human cell. For example, by altering the non-human antibodyamino acid sequence to incorporate amino acids found in human germ lineimmunoglobulin sequences. The humanized antibodies of the invention mayinclude amino acid residues not encoded by human germ lineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs. The term “humanized antibody”, as used herein, alsoincludes antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

“Hybridization,” as used herein, refers broadly to the physicalinteraction of complementary (including partially complementary)polynucleotide strands by the formation of hydrogen bonds betweencomplementary nucleotides when the strands are arranged antiparallel toeach other.

“IgV domain” and “IgC domain” as used herein, refer broadly to Igsuperfamily member domains. These domains correspond to structural unitsthat have distinct folding patterns called Ig folds. Ig folds arecomprised of a sandwich of two P sheets, each consisting of antiparallelβ strands of 5-10 amino acids with a conserved disulfide bond betweenthe two sheets in most, but not all, domains. IgC domains of Ig, TCR,and MHC molecules share the same types of sequence patterns and arecalled the cl set within the Ig superfamily. Other IgC domains fallwithin other sets. IgV domains also share sequence patter and are calledV set domains. IgV domains are longer than C-domains and form anadditional pair of β strands.

“Immune cell,” as used herein, refers broadly to cells that are ofhematopoietic origin and that play a role in the immune response. Immunecells include but are not limited to lymphocytes, such as B cells and Tcells; natural killer cells; dendritic cells, and myeloid cells, such asmonocytes, macrophages, eosinophils, mast cells, basophils, andgranulocytes.

“Immunoassay,” as used herein, refers broadly to an assay that uses anantibody to specifically bind an antigen. The immunoassay may becharacterized by the use of specific binding properties of a particularantibody to isolate, target, and/or quantify the antigen.

“Immune related disease (or disorder or condition)” as used hereinshould be understood to encompass any disease disorder or conditionselected from the group including but not limited to autoimmunediseases, inflammatory disorders and immune disorders associated withgraft transplantation rejection, such as acute and chronic rejection oforgan transplantation, allogenic stem cell transplantation, autologousstem cell transplantation, bone marrow transplantation, and graft versushost disease.

“Immune response,” as used herein, refers broadly to T cell-mediatedand/or B cell-mediated immune responses that are influenced bymodulation of T cell costimulation. Exemplary immune responses include Bcell responses (e.g., antibody production) T cell responses (e.g.,cytokine production, and cellular cytotoxicity) and activation ofcytokine responsive cells, e.g., macrophages. As used herein, the term“downmodulation” with reference to the immune response includes adiminution in any one or more immune responses, while the term“upmodulation” with reference to the immune response includes anincrease in any one or more immune responses. It will be understood thatupmodulation of one type of immune response may lead to a correspondingdownmodulation in another type of immune response. For example,upmodulation of the production of certain cytokines (e.g., IL-10) canlead to downmodulation of cellular immune responses.

“Immunologic”, “immunological” or “immune” response herein refer to thedevelopment of a humoral (antibody mediated) and/or a cellular (mediatedby antigen-specific T cells or their secretion products) responsedirected against a peptide in a recipient patient. Such a response canbe an active response induced by administration of immunogen or apassive response induced by administration of antibody or primedT-cells. Without wishing to be limited by a single hypothesis, acellular immune response is elicited by the presentation of polypeptideepitopes in association with Class II or Class I MHC molecules toactivate antigen-specific CD4⁺ T helper cells and/or CD8⁺ cytotoxic Tcells, respectively. The response may also involve activation ofmonocytes, macrophages, NK cells, basophils, dendritic cells,astrocytes, microglia cells, eosinophils, activation or recruitment ofneutrophils or other components of innate immunity. The presence of acell-mediated immunological response can be determined by proliferationassays (CD4⁺ T cells) or CTL (cytotoxic T lymphocyte) assays. Therelative contributions of humoral and cellular responses to theprotective or therapeutic effect of an immunogen can be distinguished byseparately isolating antibodies and T cells from an immunized syngeneicanimal and measuring protective or therapeutic effect in a secondsubject.

“Immunogenic agent” or “immunogen” is a moiety capable of inducing animmunological response against itself on administration to a mammal,optionally in conjunction with an adjuvant.

“Inflammatory disorders”, “inflammatory conditions” and/or“inflammation”, used interchangeably herein, refers broadly to chronicor acute inflammatory diseases, and expressly includes inflammatoryautoimmune diseases and inflammatory allergic conditions. Theseconditions include by way of example inflammatory abnormalitiescharacterized by dysregulated immune response to harmful stimuli, suchas pathogens, damaged cells, or irritants. Inflammatory disordersunderlie a vast variety of human diseases. Non-immune diseases withetiological origins in inflammatory processes include cancer,atherosclerosis, and ischemic heart disease. Examples of disordersassociated with inflammation include: Chronic prostatitis,Glomerulonephritis, Hypersensitivities, Pelvic inflammatory disease,Reperfusion injury, Sarcoidosis, Vasculitis, Interstitial cystitis,normocomplementemic urticarial vasculitis, pericarditis, myositis,anti-synthetase syndrome, scleritis, macrophage activation syndrome,Behget's Syndrome, PAPA Syndrome, Blau's Syndrome, gout, adult andjuvenile Still's disease, cryropyrinopathy, Muckle-Wells syndrome,familial cold-induced auto-inflammatory syndrome, neonatal onsetmultisystemic inflammatory disease, familial Mediterranean fever,chronic infantile neurologic, cutaneous and articular syndrome, systemicjuvenile idiopathic arthritis, Hyper IgD syndrome, Schnitzler'ssyndrome, TNF receptor-associated periodic syndrome (TRAPSP),gingivitis, periodontitis, hepatitis, cirrhosis, pancreatitis,myocarditis, vasculitis, gastritis, gout, gouty arthritis, andinflammatory skin disorders, selected from the group consisting ofpsoriasis, atopic dermatitis, eczema, rosacea, urticaria, and acne.

“Inhibitory signal,” as used herein, refers broadly to a signaltransmitted via an inhibitory receptor molecule on an immune cell. Asignal antagonizes a signal via an activating receptor (e.g., via a TCR,CD3, BCR, or Fc molecule) and can result, e.g., in inhibition of: secondmessenger generation; proliferation; or effector function in the immunecell, e.g., reduced phagocytosis, antibody production, or cellularcytotoxicity, or the failure of the immune cell to produce mediators(e.g., cytokines (e.g., IL-2) and/or mediators of allergic responses);or the development of anergy.

“Isolated,” as used herein, refers broadly to material removed from itsoriginal environment in which it naturally occurs, and thus is alteredby the hand of man from its natural environment and includes“recombinant” polypeptides. Isolated material may be, for example,exogenous nucleic acid included in a vector system, exogenous nucleicacid contained within a host cell, or any material which has beenremoved from its original environment and thus altered by the hand ofman (e.g., “isolated antibody”). For example, “isolated” or “purified,”as used herein, refers broadly to a protein, DNA, antibody, RNA, orbiologically active portion thereof, that is substantially free ofcellular material or other contaminating proteins from the cell ortissue source from which the biological substance is derived, orsubstantially free from chemical precursors or other chemicals whenchemically synthesized. As used herein the term “isolated” refers to acompound of interest (for example a polynucleotide or a polypeptide)that is in an environment different from that in which the compoundnaturally occurs e.g., separated from its natural milieu such as byconcentrating a peptide to a concentration at which it is not found innature. “Isolated” includes compounds that are within samples that aresubstantially enriched for the compound of interest and/or in which thecompound of interest is partially or substantially purified.

“Isolated antibody”, as used herein, is intended to refer to an antibodythat is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds VISTA) is substantially free of antibodies that specifically bindantigens other than VISTA). Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

“Isotype” herein refers to the antibody class (e.g., IgM or IgGI) thatis encoded by the heavy chain constant region genes.

“K-assoc” or “Ka”, as used herein, refers broadly to the associationrate of a particular antibody-antigen interaction, whereas the term“Kdiss” or “Kd,” as used herein, refers to the dissociation rate of aparticular antibody-antigen interaction.

The term “K_(D)”, as used herein, is intended to refer to thedissociation constant, which is obtained from the ratio of Kd to Ka(i.e., Kd/Ka) and is expressed as a molar concentration (M). K_(D)values for antibodies can be determined using methods well establishedin the art such as plasmon resonance (BIAcore®), ELISA and KINEXA. Apreferred method for determining the K_(D) of an antibody is by usingsurface Plasmon resonance, preferably using a biosensor system such as aBIAcore® system or by ELISA. Typically these methods are effected at 25°or 37° C. Antibodies for therapeutic usage generally will possess aK_(D) when determined by surface Plasmon resonance of 50 nM or less ormore typically I nM or less at 250 or 37° C.

“Label” or a “detectable moiety” as used herein, refers broadly to acomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, chemical, or other physical means.

“Low stringency,” “medium stringency,” “high stringency,” or “very highstringency conditions,” as used herein, refers broadly to conditions fornucleic acid hybridization and washing. Guidance for performinghybridization reactions can be found in Ausubel, et al., Short Protocolsin Molecular Biology (5th Ed.) John Wiley & Sons, NY (2002). Exemplaryspecific hybridization conditions include but are not limited to: (1)low stringency hybridization conditions in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1%SDS at least at 50° C. (the temperature of the washes can be increasedto 55° C. for low stringency conditions); (2) medium stringencyhybridization conditions in 6×SSC at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 60° C.; (3) high stringencyhybridization conditions in 6×SSC at about 45° C. followed by one ormore washes in 0.2×. SSC, 0.1% SDS at 65° C.; and (4) very highstringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at65° C., followed by one or more washes at 0.2×SSC, and 1% SDS at 65° C.

“Mammal,” as used herein, refers broadly to any and all warm-bloodedvertebrate animals of the class Mammalia, including humans,characterized by a covering of hair on the skin and, in the female,milk-producing mammary glands for nourishing the you ng. Examples ofmammals include but are not limited to alpacas, armadillos, capybaras,cats, camels, chimpanzees, chinchillas, cattle, dogs, goats, gorillas,hamsters, horses, humans, lemurs, llamas, mice, non-human primates,pigs, rats, sheep, shrews, squirrels, tapirs, and voles. Mammals includebut are not limited to bovine, canine, equine, feline, murine, ovine,porcine, primate, and rodent species. Mammal also includes any and allthose listed on the Mammal Species of the World maintained by theNational Museum of Natural History, Smithsonian Institution inWashington D.C.

“Multispecific antibody” refers to an antibody with 2 or more antigenbinding regions. This includes bispecific antibodies. These antigenbinding regions may bind to different antigens or to different epitopesof the same antigen.

“Naturally-occurring nucleic acid molecule,” as used herein, refersbroadly refers to an RNA or DNA molecule having a nucleotide sequencethat occurs in nature (e.g., encodes a natural protein).

“Nucleic acid” or “nucleic acid sequence,” as used herein, refersbroadly to a deoxy-ribonucleotide or ribonucleotide oligonucleotide ineither single- or double-stranded form. The term encompasses nucleicacids, i.e., oligonucleotides, containing known analogs of naturalnucleotides. The term also encompasses nucleic-acid-like structures withsynthetic backbones. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions) andcomplementary sequences, as well as the sequence explicitly indicated.The term nucleic acid is used interchangeably with gene, cDNA, m RNA,oligonucleotide, and polynucleotide.

“Operatively linked”, as used herein, refers broadly to when two DNAfragments are joined such that the amino acid sequences encoded by thetwo DNA fragments remain in-frame.

“Paratope,” as used herein, refers broadly to the part of an antibodywhich recognizes an antigen (e.g., the antigen-binding site of anantibody.) Paratopes may be a small region (e.g., 15-22 amino acids) ofthe antibody's Fv region and may contain parts of the antibody's heavyand light chains. See Goldsby, et al. Antigens (Chapter 3) Immunology(5th Ed.) New York: W. H. Freeman and Company, pages 57-75.

“Patient,” or “subject” or “recipient”, “individual”, or “treatedindividual” are used interchangeably herein, and refers broadly to anyanimal that is in need of treatment either to alleviate a disease stateor to prevent the occurrence or reoccurrence of a disease state. Also,“Patient” as used herein, refers broadly to any animal that has riskfactors, a history of disease, susceptibility, symptoms, and signs, waspreviously diagnosed, is at risk for, or is a member of a patientpopulation for a disease. The patient may be a clinical patient such asa human or a veterinary patient such as a companion, domesticated,livestock, exotic, or zoo animal.

“Polypeptide,” “peptide” and “protein,” are used interchangeably andrefer broadly to a polymer of amino acid residues s of any length,regardless of modification (e.g., phosphorylation or glycosylation). Theterms apply to amino acid polymers in which one or more amino acidresidue is an analog or mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer. Polypeptidescan be modified, e.g., by the addition of carbohydrate residues to formglycoproteins. The terms “polypeptide,” “peptide” and “protein”expressly include glycoproteins, as well as non-glycoproteins.

“Promoter,” as used herein, refers broadly to an array of nucleic acidsequences that direct transcription of a nucleic acid. As used herein, apromoter includes necessary nucleic acid sequences near the start siteof transcription, such as, in the case of a polymerase II type promoter,a TATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation.

“Prophylactically effective amount,” as used herein, refers broadly tothe amount of a compound that, when administered to a patient forprophylaxis of a disease or prevention of the reoccurrence of a disease,is sufficient to effect such prophylaxis for the disease orreoccurrence. The prophylactically effective amount may be an amounteffective to prevent the incidence of signs and/or symptoms. The“prophylactically effective amount” may vary depending on the diseaseand its severity and the age, weight, medical history, predisposition toconditions, preexisting conditions, of the patient to be treated.

“Prophylactic vaccine” and/or “Prophylactic vaccination” refers to avaccine used to prevent a disease or symptoms associated with a diseasesuch as cancer or an infectious condition.

“Prophylaxis,” as used herein, refers broadly to a course of therapywhere signs and/or symptoms are not present in the patient, are inremission, or were previously present in a patient. Prophylaxis includespreventing disease occurring subsequent to treatment of a disease in apatient. Further, prevention includes treating patients who maypotentially develop the disease, especially patients who are susceptibleto the disease (e.g., members of a patent population, those with riskfactors, or at risk for developing the disease).

“Recombinant” as used herein, refers broadly with reference to aproduct, e.g., to a cell, or nucleic acid, protein, or vector, indicatesthat the cell, nucleic acid, protein or vector, has been modified by theintroduction of a heterologous nucleic acid or protein or the alterationof a native nucleic acid or protein, or that the cell is derived from acell so modified. Thus, for example, recombinant cells express genesthat are not found within the native (non-recombinant) form of the cellor express native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further below), (b)antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germ lineimmunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies can be subjected to in vitro mutagenesis(or, when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germ line VH and VL sequences, may notnaturally exist within the human antibody germ line repertoire in vivo.

“Signal sequence” or “signal peptide,” as used herein, refers broadly toa peptide containing about 15 or more amino acids which occurs at theN-terminus of secretory and membrane bound polypeptides and whichcontains a large number of hydrophobic amino acid residues. For example,a signal sequence contains at least about 10-30 amino acid residues,preferably about 15-25 amino acid residues, more preferably about 18-20amino acid residues, and even more preferably about 19 amino acidresidues, and has at least about 35-65%, preferably about 38-50%, andmore preferably about 40-45% hydrophobic amino acid residues (e.g.,Valine, Leucine, Isoleucine or Phenylalanine). A “signal sequence,” alsoreferred to in the art as a “signal peptide,” serves to direct apolypeptide containing such a sequence to a lipid bilayer, and iscleaved in secreted.

“Specifically (or selectively) binds” to an antibody or “specifically(or selectively) immunoreactive with,” or “specifically interacts orbinds,” as used herein, refers broadly to a protein or peptide (or otherepitope), refers, in some embodiments, to a binding reaction that isdeterminative of the presence of the protein in a heterogeneouspopulation of proteins and other biologies. For example, underdesignated immunoassay conditions, the specified antibodies bind to aparticular protein at least two times greater than the background(non-specific signal) and do not substantially bind in a significantamount to other proteins present in the sample. Typically a specific orselective reaction will be at least twice background signal or noise andmore typically more than about 10 to 100 times background.

“Specifically hybridizable” and “complementary” as used herein, referbroadly to a nucleic acid can form hydrogen bond(s) with another nucleicacid sequence by either traditional Watson-Crick or othernon-traditional types. The binding free energy for a nucleic acidmolecule with its complementary sequence is sufficient to allow therelevant function of the nucleic acid to proceed, e.g., RNAi activity.Determination of binding free energies for nucleic acid molecules iswell known in the art. (See, e.g., Turner, et al. CSH Symp. Quant. Biol.LII: 123-33 (1987); Frier, et al. PNAS 83: 9373-77 1986); Turner, et al.J. Am. Chem. Soc. 109:3783-85 (1987)). A percent complementarityindicates the percentage of contiguous residues in a nucleic acidmolecule that can form hydrogen bonds (e.g., Watson-Crick base pairing)with a second nucleic acid sequence (e.g., about at least 5, 6, 7, 8, 9,10 out of 10 being about at least 50%, 60%, 70%, 80%, 90%, and 100%complementary, inclusive). “Perfectly complementary” or 100%complementarity refers broadly all of the contiguous residues of anucleic acid sequence hydrogen bonding with the same number ofcontiguous residues in a second nucleic acid sequence.

“Substantial complementarity” refers to polynucleotide strandsexhibiting about at least 90% complementarity, excluding regions of thepolynucleotide strands, such as overhangs, that are selected so as to benoncomplementary. Specific binding requires a sufficient degree ofcomplementarity to avoid non-specific binding of the oligomeric compoundto non-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, or in the case of in vitro assays,under conditions in which the assays are performed. The non-targetsequences typically may differ by at least 5 nucleotides.

“Signs” of disease, as used herein, refers broadly to any abnormalityindicative of disease, discoverable on examination of the patient; anobjective indication of disease, in contrast to a symptom, which is asubjective indication of disease.

“Solid support,” “support,” and “substrate,” as used herein, refersbroadly to any material that provides a solid or semi-solid structurewith which another material can be attached including but not limited tosmooth supports (e.g., metal, glass, plastic, silicon, and ceramicsurfaces) as well as textured and porous materials.

“Soluble ectodomain (ECD)” or “ectodomain” or “soluble VISTAprotein(s)/molecule(s)” of VISTA as used herein meansnon-cell-surface-bound VISTA molecules or any portion thereof,including, but not limited to: VISTA fusion proteins or VISTA ECD-Igfusion proteins, wherein the extracellular domain of VISTA or fragmentthereof is fused to an immunoglobulin (Ig) moiety rendering the fusionmolecule soluble, or fragments and derivatives thereof, proteins withthe extracellular domain of VISTA fused or joined with a portion of abiologically active or chemically active protein such as thepapillomavirus E7 gene product, melanoma-associated antigen p97 or HIVenv protein, or fragments and derivatives thereof; hybrid (chimeric)fusion proteins such as VISTA-Ig, or fragments and derivatives thereof.Such fusion proteins are described in greater detail below.

“Soluble VISTA protein(s)/molecule(s)” herein also include VISTAmolecules with the transmembrane domain removed to render the proteinsoluble, or fragments and derivatives thereof; fragments, portions orderivatives thereof, and soluble VISTA mutant molecules. The solubleVISTA molecules used in the methods according to at least someembodiments of the invention may or may not include a signal (leader)peptide sequence.

“Subject” or “patient” or “individual” in the context of therapy ordiagnosis herein includes any human or non human animal. The term“nonhuman animal” includes all vertebrates, e.g., mammals andnon-mammals, such as non human primates, sheep, dogs, cats, horses,cows, chickens, amphibians, reptiles, etc., i.e., anyone suitable to betreated according to the present invention include, but are not limitedto, avian and mammalian subjects, and are preferably mammalian. Anymammalian subject in need of being treated according to the presentinvention is suitable. Human subjects of both genders and at any stageof development (i.e., neonate, infant, juvenile, adolescent, and adult)can be treated according to the present invention. The present inventionmay also be carried out on animal subjects, particularly mammaliansubjects such as mice, rats, dogs, cats, cattle, goats, sheep, andhorses for veterinary purposes, and for drug screening and drugdevelopment purposes. “Subjects” is used interchangeably with“individuals” and “patients.”

“Substantially free of chemical precursors or other chemicals,” as usedherein, refers broadly to preparations of VISTA protein in which theprotein is separated from chemical precursors or other chemicals whichare involved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of VISTA protein having less than about 30% (bydry weight) of chemical precursors or non-VISTA chemicals, morepreferably less than about 20% chemical precursors or non-VISTAchemicals, still more preferably less than about 10% chemical precursorsor non-VISTA chemicals, and most preferably less than about 5% chemicalprecursors or non-VISTA chemicals.

“Symptoms” of disease as used herein, refers broadly to any morbidphenomenon or departure from the normal in structure, function, orsensation, experienced by the patient and indicative of disease.

“T cell,” as used herein, refers broadly to CD4+ T cells and CD8+ Tcells. The term T cell also includes both T helper 1 type T cells and Thelper 2 type T cells.

“Therapy,” “therapeutic,” “treating,” or “treatment”, as used herein,refers broadly to treating a disease, arresting, or reducing thedevelopment of the disease or its clinical symptoms, and/or relievingthe disease, causing regression of the disease or its clinical symptoms.Therapy encompasses prophylaxis, treatment, remedy, reduction,alleviation, and/or providing relief from a disease, signs, and/orsymptoms of a disease. Therapy encompasses an alleviation of signsand/or symptoms in patients with ongoing disease signs and/or symptoms(e.g., inflammation, pain). Therapy also encompasses “prophylaxis”. Theterm “reduced”, for purpose of therapy, refers broadly to the clinicalsignificant reduction in signs and/or symptoms. Therapy includestreating relapses or recurrent signs and/or symptoms (e.g.,inflammation, pain). Therapy encompasses but is not limited toprecluding the appearance of signs and/or symptoms anytime as well asreducing existing signs and/or symptoms and eliminating existing signsand/or symptoms. Therapy includes treating chronic disease(“maintenance”) and acute disease. For example, treatment includestreating or preventing relapses or the recurrence of signs and/orsymptoms (e.g., inflammation, pain).

“Treg cell” (sometimes also referred to as suppressor T cells orinducible Treg cells or iTregs) as used herein refers to a subpopulationof T cells which modulate the immune system and maintain tolerance toself-antigens and can abrogate autoimmune diseases. Foxp3⁺ CD4⁺CD25⁺regulatory T cells (Tregs) are critical in maintaining peripheraltolerance under normal conditions.

“Transmembrane domain,” as used herein, refers broadly to an amino acidsequence of about 15 amino acid residues in length which spans theplasma membrane. More preferably, a transmembrane domain includes aboutat least 20, 25, 30, 35, 40, or 45 amino acid residues and spans theplasma membrane. Transmembrane domains are rich in hydrophobic residues,and typically have an a-helical structure. In an embodiment, at least50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of atransmembrane domain are hydrophobic, e.g., leucines, isoleucines,tyrosines, or tryptophans. Transmembrane domains are described in, forexample, Zagotta, et al. Annu. Rev. Neurosci. 19:235-263 (1996).

“Transgenic animal,” as used herein, refers broadly to a non-humananimal, preferably a mammal, more preferably a mouse, in which one ormore of the cells of the animal includes a “transgene”. The term“transgene” refers to exogenous DNA which is integrated into the genomeof a cell from which a transgenic animal develops and which remains inthe genome of the mature animal, for example directing the expression ofan encoded gene product in one or more cell types or tissues of thetransgenic animal.

“Unresponsiveness,” as used herein, refers broadly to refractivity ofimmune cells to stimulation, e.g., and stimulation via an activatingreceptor or a cytokine. Unresponsiveness can occur, e.g., because ofexposure to immunosuppressants or high doses of antigen.

“Variable region” or “VR,” as used herein, refers broadly to the domainswithin each pair of light and heavy chains in an antibody that areinvolved directly in binding the antibody to the antigen. Each heavychain has at one end a variable domain (V_(H)) followed by a number ofconstant domains. Each light chain has a variable domain (V_(L)) at oneend and a constant domain at its other end; the constant domain of thelight chain is aligned with the first constant domain of the heavychain, and the light chain variable domain is aligned with the variabledomain of the heavy chain.

“Vector,” as used herein, refers broadly to a nucleic acid moleculecapable of transporting another nucleic acid molecule to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Vectors are referred to herein as “recombinant expression vectors” orsimply “expression vectors”. In general, expression vectors of utilityin recombinant DNA techniques are often in the form of plasmids. In thepresent specification, “plasmid” and “vector” may be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions. The techniques and procedures are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification. See, e.g., Sambrook,et al. Molec. Cloning: Lab. Manual [3rd Ed] Cold Spring HarborLaboratory Press (2001). Standard techniques may be used for recombinantDNA, oligonucleotide synthesis, and tissue culture, and transformation(e.g., electroporation, lipofection). Enzymatic reactions andpurification techniques may be performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein.

Having defined certain terms and phrases used in the presentapplication, the anti-VISTA antibodies and antigen binding antibodyfragments and methods for the production and use thereof which areembraced by the invention are further described below.

The present invention relates to antibodies and antibody fragmentscomprising an antigen binding region that binds to a V-domain IgSuppressor of T cell Activation (VISTA). VISTA is a checkpoint regulatorthat negatively suppresses immune responses. See Wang et al., “VISTA, anovel mouse Ig superfamily ligand that negatively regulates T cellresponses,” J. Exp. Med., 208(3) 577-92 (2011). This protein isexpressed on normal human neutrophils, monocytes and T cells subsets. Inaddition, cynomolgus monkey cells express VISTA in a similar pattern tonormal human cells. VISTA is also expressed in the peripheral bloodcells e.g., of cancer patients.

The binding of an antagonist anti-VISTA antibody or antibody fragment toVISTA according to the invention will antagonize at least one of theeffects of VISTA on immunity thereby suppressing the suppressive effectsof VISTA on immunity, e.g., T cell immunity and/or cytokine expression.By contrast, the binding of an agonist anti-VISTA antibody or antibodyfragment to VISTA according to the invention will agonize, elicit ormimic at least one of the effects of VISTA on immunity thereby promotingat least one of the suppressive effects of VISTA on immunity, e.g., thesuppression of T cell immunity or the suppression of the expression ofspecific proinflammatory cytokines or its promoting effect on theexpression of certain chemoattractants and chemokines.

Such antibody fragments include by way of example Fab, F(ab′)₂, and scFvantibody fragments. These antibody or antibody fragments can comprise anantibody constant region or fragment or variant thereof. Such antibodiesand antibody fragments include those which bind to VISTA proteinsexpressed on hematopoietic and other cells, for example, myeloid cellsand/or lymphocytes, monocytes, neutrophils, T cells, natural killer (NK)cells, natural killer T (NKT) cells, a tumor cell, and/or in the tumormicroenvironment (TME). The tumor microenvironment is the cellularenvironment of the tumor. It can include surrounding immune cells,fibroblasts, blood vessels, other cells, signaling molecules, and theextracellular matrix.

Antibodies that block or inhibit the effects of VISTA may be used toenhance human immune responses, in particular immune responses tomalignancies and infection. By contrast molecules that agonize VISTAsuch as soluble VISTA, e.g., VISTA-Ig and the subject agonist anti-humanVISTA antibodies and fragments may be used to suppress undesired humanimmune responses such as autoimmune, allergic, GVHD, sepsis orundesirable inflammatory immune responses.

The subject application provides novel antagonist and agonist anti-humanVISTA antibodies including those comprising the same CDRS as any of theanti-human VISTA antibodies having the sequences shown in FIG. 4A-4JJ.While prior to the present invention a number of antagonist anti-humanVISTA antibodies have been reported in the literature, no agonisticanti-human VISTA antibodies or antibody fragments have been reported.

As disclosed in the experimental examples which follow the inventorsinitially produced 2 chimeric anti-human VISTA antibodies derived from amurine anti-human VISTA antibody (1E8 having sequences in FIG. 4A-4JJ)which respectively contain unmodified IgG2 human constant regions orIgG2 constant regions wherein the cysteine residue at position 127 ofthe kappa chain was changed to a serine residue. As shown in theExamples and the Figures referenced therein, both antibodies were foundto agonize or mimic the suppressive effects of VISTA on immunity atleast based on (i) their ability to decrease the expression of certainproinflammatory cytokines such as IL-2, IL-4, IL-6, IL-17, granulocytemacrophage colony stimulating factor (GM-CSF) and tumor necrosisfactor-alpha (TNF-α) as well as reducing the expression of certainchemokines or chemoattractants such as KC (keratinocyte derivedchemokine) or MIP-2 (Macrophage Inflammatory Protein-2); (ii) suppress Tcell activity in GVHD model; and to (iii) suppress CD3-driven T cellresponses.

Additionally after isolation of these 2 agonist antibodies another 10chimeric agonist anti-human VISTA antibodies containing human IgG2constant or Fc regions have been obtained using analogous methods. Theseantibodies were derived from the antibodies referred to herein as GG8,VSTB95 (INX903), VSTB103 (INX904), VSTB53 (INX905), VSTB92 (INX908),VSTB50 (INX900), VSTB56 (INX901), VSTB63 (INX902), VSTB54 (INX906) andVSTB66 (INX907)(having the sequences in FIG. 4A-4JJ).

Particularly, these chimeric anti-human VISTA antibodies have thevariable sequences shown in FIG. 4A-4JJ and human IgG2 constant regions.As reported in the Summary Tables 1 and 2 infra these anti-human VISTAantibodies when assessed by use of antibody binning were found to bindto 2 different epitope groups designated Group 1 and Group 2. As notedin FIG. 4A-4JJ the epitope corresponding to Group 2 includes residues in2 different peptides present in human VISTA, i.e., NLTLLDSGL andVQTGKDAPSNC.

As is indicated in the Tables 1 and 2 infra these 12 differentanti-human VISTA antibodies were found to be immunosuppressive in atleast one model of immunosuppression and many in severalimmunosuppression models. Particularly INX905, INX908, INX901, INX902and INX906 were shown to be immunosuppressive in 2 different assayformats. While all of these antibodies were immunosuppressive and appearto elicit, promote or agonize the immunosuppressive effects of VISTA,INX901, INX902 and INX906 and INX908 appear to be the mostimmunosuppressive.

Also, other chimeric anti-human VISTA antibodies comprising human IgG2constant domains containing the variable sequences of other anti-VISTAantibodies shown in FIG. 4A-4JJ are to be screened for theirimmunosuppressive properties and their ability to agonize or mimic theimmunosuppressive and other effects of human VISTA. Based on the resultsobtained to date this screening should identify other agonist anti-humanVISTA antibodies, particularly those which bind the same epitope.Additionally agonist anti-human VISTA antibodies according to theinvention have been shown to be effective (immunosuppressive) innumerous autoimmune and inflammatory animal disease models includingarthritis, lupus or SLE, GVHD, inflammatory bowel disease (IBD) orcolitis, chronic and acute infectious disease or hepatotoxicity andpsoriasis animal models. Based thereon the subject anti-human VISTAagonist antibodies should be well suited for use in therapeutic andprophylactic treatment of autoimmune, allergic and inflammatoryconditions.

As noted chimeric IgG2 anti-human VISTA antibodies having the sequencesshown in FIG. 4A-4JJ were shown to be immunosuppressive in differentmodels of immunosuppression. These antibodies moreover elicit theseimmunosuppressive effects in a specific immunomodulatory manner ratherthan by effecting the depletion of specific types of T cells or bydepleting T cells in general.

As further shown in the examples chimeric IgG2 agonistic anti-humanVISTA antibodies containing a mutation in the hinge region elicitedsubstantially the same suppressive effects on immunity, i.e., themutation within in IgG2 constant region appeared to elicit noenhancement in suppression under the tested experimental conditions.Rather both the IgG2A and IgG2 B forms and mixtures thereof elicited thesame immunosuppressive effects. Additionally, based on experimentsdisclosed in the examples it would appear that FcyR binding maycontribute to the agonist properties of the subject anti-human VISTAantibodies. In particular it was found that the inclusion of silent IgG2constant regions ablated the immunosuppressive properties of the testedagonist antibodies. Based on these results it is hypothesized that oneor more FcyRs may affect the agonistic properties of these antibodiesand in particular it is hypothesized that FcyRIIA (CD32 or CD32A) orFcyRIIB (CD32B) binding may be involved in the agonist properties of thesubject agonist antibodies.

Using these same methods it is expected that other agonist anti-humanVISTA IgG2 antibodies may be obtained, e.g., others derived fromanti-human VISTA antibodies having the sequences shown in FIG. 4A-4JJ.As mentioned 12 agonist anti-human VISTA antibodies have been obtainedto date including those having the sequences contained in FIG. 4A-4JJ.Based on these results it is anticipated that other agonistic anti-humanVISTA antibodies may be generated and shown to be immunosuppressive.Also it is anticipated that other agonistic anti-human VISTA antibodiesmay be generated which bind to the same or overlapping epitope and/orcompete with any of the antibodies containing the sequences shown inFIG. 4A-4JJ. In exemplary embodiments these antibodies will bind to theepitope corresponding to Group 1 or Group 2 antibodies or will competefor binding to human VISTA with such antibodies.

Methods for identifying the specific epitope(s) bound by an antibody areknown in the art. In the working examples Applicants disclose theelucidation of the epitope bound by a number of anti-VISTA antibodiesaccording to the invention. Thus, in exemplary embodiments agonistanti-human VISTA antibodies according to the invention will compriseIgG2 constant regions or fragments thereof, of the A form, B form or amixture of the foregoing. In exemplary embodiments these antibodies willbind to one or more FcyRs, e.g., they will bind to the same FcyRs as anintact or wild-type human IgG2 Fc region. In other exemplary embodimentsthe antibody will bind to CD32 (CD32A and/or CD32B). This may beaccomplished by the use of wild-type or modified IgG2 constant regionswhich bind to CD32 (CD32A and/or CD32B). Further, the agonist antibodymay be modified to incorporate another polypeptide such as another Fcpolypeptide or antigen binding region which binds to FcyRs such as CD32Aand/or CD32B.

The IgG2 Fc or constant regions contained in the inventive agonistanti-human VISTA antibodies optionally may be modified, e.g., in orderto alter effector function, e.g., to alter FcR binding, FcRN binding,complement binding, glycosylation and the like. In particular, the IgG2Fc or constant regions contained in the inventive agonist anti-humanVISTA antibodies optionally may be modified by the conversion of thecysteine at position 27 or further optionally by the conversion ofanother cysteine residue or other residues, e.g., in the hinge region toanother amino acid, e.g., a serine. Other potential Fc modifications aredisclosed infra.

These VISTA agonist antibodies may be used in treating or preventingdiseases conditions or for treating or reducing, ameliorating thepathological effects associated therewith, e.g., inflammation, intreating or preventing conditions wherein the suppression of T cellimmunity or the expression of proinflammatory cytokines and or increasedexpression of chemokines and chemoattractants is therapeutically orprophylactically beneficial. These conditions include in particularautoimmunity, allergy, inflammatory disorders, sepsis, GVHD and forinhibiting unwanted T cell immune responses against transplanted cells,tissues or organs such as CAR-T cell or gene therapy constructs or cellscontaining.

As mentioned exemplary conditions which may be treated therapeuticallyor prophylactically using an agonist anti-human VISTA antibody accordingto the invention include autoimmune conditions, allergy conditions,inflammatory conditions, GVHD, transplant and sepsis. As mentioned,agonist anti-human VISTA antibodies according to the invention have beenshown to be therapeutically effective and to be immunosuppressive innumerous animal disease models including arthritis, inflammatory boweldisease (IBD), lupus, GVHD, chronic acute infection/hepatotoxicity andpsoriasis disease models. Therefore the inventive antibodies should bewell suited for use in treating conditions wherein the suppression ofimmunity, especially T cell immunity is therapeutically desired.

A. Use of Agonistic or Antagonistic Anti-Human Vista Antibodies andFragments in Therapy and Diagnosis

Compositions containing agonists according to the invention may be usedto inhibit T cell immunity and to treat conditions where this istherapeutically desirable such as autoimmunity, allergy or inflammatoryconditions. These compositions will comprise an amount of an agonistantibody or antibody fragment according to the invention effective tosuppress T cell activation or proliferation or cytokine expression orother effects of VISTA in a subject in need thereof. Such autoimmune,inflammatory and allergic conditions include for example arthriticconditions such as RA, psoriatic arthritis, psoriasis, scleroderma,multiple sclerosis, lupus, IBD, ITP, diabetes, GVHD, sarcoidosis,allergic asthma, hepatitis associated hepatotoxicity and for inhibitingunwanted T cell immune responses against transplanted cells, tissues ororgans such as CAR-T cell or gene therapy constructs or cells containingand the like.

Specific conditions wherein the inventive antibodies may be used aloneor in association with other therapeutics, especially otherimmunosuppressant molecules include acquired immune deficiency syndrome(AIDS), acquired splenic atrophy, acute anterior uveitis, AcuteDisseminated Encephalomyelitis (ADEM), acute gouty arthritis, acutenecrotizing hemorrhagic leukoencephalitis, acute or chronic sinusitis,acute purulent meningitis (or other central nervous system inflammatorydisorders), acute serious inflammation, Addison's disease, adrenalitis,adult onset diabetes mellitus (Type II diabetes), adult-onset idiopathichypoparathyroidism (AOIH), Agammaglobulinemia, agranulocytosis,vasculitides, including vasculitis, optionally, large vessel vasculitis,optionally, polymyalgia rheumatica and giant cell (Takayasu's)arthritis, allergic conditions, allergic contact dermatitis, allergicdermatitis, allergic granulomatous angiitis, allergic hypersensitivitydisorders, allergic neuritis, allergic reaction, alopecia areata,alopecia totalis, Alport's syndrome, alveolitis, optionally allergicalveolitis or fibrosing alveolitis, Alzheimer's disease, amyloidosis,amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), aneosinophil-related disorder, optionally eosinophilia, anaphylaxis,ankylosing spondylitis, angiectasis, antibody-mediated nephritis,Anti-GBM/Anti-TBM nephritis, antigen-antibody complex-mediated diseases,antiglomerular basement membrane disease, anti-phospholipid antibodysyndrome, antiphospholipid syndrome (APS), aphthae, aphthous stomatitis,aplastic anemia, arrhythmia, arteriosclerosis, arterioscleroticdisorders, arthritis, optionally rheumatoid arthritis such as acutearthritis, or chronic rheumatoid arthritis, arthritis chronicaprogrediente, arthritis deformans, ascariasis, aspergilloma, granulomascontaining eosinophils, aspergillosis, aspermiogenese, asthma,optionally asthma bronchiale, bronchial asthma, or auto-immune asthma,ataxia telangiectasia, ataxic sclerosis, atherosclerosis, autism,autoimmune angioedema, autoimmune aplastic anemia, autoimmune atrophicgastritis, autoimmune diabetes, autoimmune disease of the testis andovary including autoimmune orchitis and oophoritis, autoimmune disordersassociated with collagen disease, autoimmune dysautonomia, autoimmuneear disease, optionally autoimmune inner ear disease (AGED), autoimmuneendocrine diseases including thyroiditis such as autoimmune thyroiditis,autoimmune enteropathy syndrome, autoimmune gonadal failure, autoimmunehearing loss, autoimmune hemolysis, Autoimmune hepatitis, autoimmunehepatological disorder, autoimmune hyperlipidemia, autoimmuneimmunodeficiency, autoimmune inner ear disease (AIED), autoimmunemyocarditis, autoimmune neutropenia, autoimmune pancreatitis, autoimmunepolyendocrinopathies, autoimmune polyglandular syndrome type I,autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP),autoimmune thyroid disease, autoimmune urticaria, autoimmune-mediatedgastrointestinal diseases, Axonal & neuronal neuropathies, Balo disease,Behcet's disease, benign familial and ischemia-reperfusion injury,benign lymphocytic angiitis, Berger's disease (IgA nephropathy),bird-fancier's lung, blindness, Boeck's disease, bronchiolitisobliterans (non-transplant) vs NSIP, bronchitis, bronchopneumonicaspergillosis, Bruton's syndrome, bullous pemphigoid, Caplan's syndrome,Cardiomyopathy, cardiovascular ischemia, Castleman's syndrome, Celiacdisease, celiac sprue (gluten enteropathy), cerebellar degeneration,cerebral ischemia, and disease accompanying vascularization, Chagasdisease, channelopathies, optionally epilepsy, channelopathies of theCNS, chorioretinitis, choroiditis, an autoimmune hematological disorder,chronic active hepatitis or autoimmune chronic active hepatitis, chroniccontact dermatitis, chronic eosinophilic pneumonia, chronic fatiguesyndrome, chronic hepatitis, chronic hypersensitivity pneumonitis,chronic inflammatory arthritis, Chronic inflammatory demyelinatingpolyneuropathy (CIDP), chronic intractable inflammation, chronicmucocutaneous candidiasis, chronic neuropathy, optionally IgMpolyneuropathies or IgM-mediated neuropathy, chronic obstructive airwaydisease, chronic pulmonary inflammatory disease, Chronic recurrentmultifocal osteomyelitis (CRMO), chronic thyroiditis (Hashimoto'sthyroiditis) or subacute thyroiditis, Churg-Strauss syndrome,cicatricial pemphigoid/benign mucosal pemphigoid, CNS inflammatorydisorders, CNS vasculitis, Coeliac disease, Cogan's syndrome, coldagglutinin disease, colitis polyposa, colitis such as ulcerativecolitis, colitis ulcerosa, collagenous colitis, conditions involvinginfiltration of T cells and chronic inflammatory responses, congenitalheart block, congenital rubella infection, Coombs positive anemia,coronary artery disease, Coxsackie myocarditis, CREST syndrome(calcinosis, Raynaud's phenomenon), Crohn's disease, cryoglobulinemia,Cushing's syndrome, cyclitis, optionally chronic cyclitis, heterochroniccyclitis, iridocyclitis, or Fuch's cyclitis, cystic fibrosis,cytokine-induced toxicity, deafness, degenerative arthritis,demyelinating diseases, optionally autoimmune demyelinating diseases,demyelinating neuropathies, dengue, dermatitis herpetiformis and atopicdermatitis, dermatitis including contact dermatitis, dermatomyositis,dermatoses with acute inflammatory components, Devic's disease(neuromyelitis optica), diabetic large-artery disorder, diabeticnephropathy, diabetic retinopathy, Diamond Blackfan anemia, diffuseinterstitial pulmonary fibrosis, dilated cardiomyopathy, discoid lupus,diseases involving leukocyte diapedesis, Dressler's syndrome,Dupuytren's contracture, echovirus infection, eczema including allergicor atopic eczema, encephalitis such as Rasmussen's encephalitis andlimbic and/or brainstem encephalitis, encephalomyelitis, optionallyallergic encephalomyelitis or encephalomyelitis allergica andexperimental allergic encephalomyelitis (EAE), endarterial hyperplasia,endocarditis, endocrine ophthalmopathy, endometriosis, endomyocardialfibrosis, endophthalmia phacoanaphylactica, endophthalmitis, enteritisallergica, eosinophilia-myalgia syndrome, eosinophilic fascitis,epidemic keratoconjunctivitis, epidermolysis bullosa acquisita (EBA),episclera, episcleritis, Epstein-Barr virus infection, erythema elevatumet diutinum, erythema multiforme, erythema nodosum leprosum, erythemanodosum, erythroblastosis fetalis, esophageal dysmotility, Essentialmixed cryoglobulinemia, ethmoid, Evan's syndrome, Experimental AllergicEncephalomyelitis (EAE), Factor VIII deficiency, farmer's lung, febrisrheumatica, Felty's syndrome, fibromyalgia, fibrosing alveolitis,filariasis, focal segmental glomerulosclerosis (FSGS), food poisoning,frontal, gastric atrophy, giant cell arthritis (temporal arthritis),giant cell hepatitis, giant cell polymyalgia, glomerulonephritides,glomerulonephritis (GN) with and without nephrotic syndrome such aschronic or acute glomerulonephritis (e.g., primary GN), Goodpasture'ssyndrome, gouty arthritis, granulocyte transfusion-associated syndromes,granulomatosis including lymphomatoid granulomatosis, granulomatosiswith polyangiitis (GPA), granulomatous uveitis, Grave's disease,Guillain-Barre syndrome, gutatte psoriasis, hemoglobinuriaparoxysmatica, Hamman-Rich's disease, Hashimoto's disease, Hashimoto'sencephalitis, Hashimoto's thyroiditis, hemochromatosis, hemolytic anemiaor immune hemolytic anemia including autoimmune hemolytic anemia (AIHA),hemolytic anemia, hemophilia A, Henoch-Schonlein purpura, Herpesgestationis, human immunodeficiency virus (HIV) infection, hyperalgesia,hypogammaglobulinemia, hypogonadism, hypoparathyroidism, idiopathicdiabetes insipidus, idiopathic facial paralysis, idiopathichypothyroidism, idiopathic IgA nephropathy, idiopathic membranous GN oridiopathic membranous nephropathy, idiopathic nephritic syndrome,idiopathic pulmonary fibrosis, idiopathic sprue, Idiopathicthrombocytopenic purpura (ITP), IgA nephropathy, IgE-mediated diseases,optionally anaphylaxis and allergic or atopic rhinitis, IgG4-relatedsclerosing disease, ileitis regionalis, immune complex nephritis, immuneresponses associated with acute and delayed hypersensitivity mediated bycytokines and T-lymphocytes, immune-mediated GN, immunoregulatorylipoproteins, including adult or acute respiratory distress syndrome(ARDS), Inclusion body myositis, infectious arthritis, infertility dueto antispermatozoan antibodies, inflammation of all or part of the uvea,inflammatory bowel disease (IBD) inflammatory hyperproliferative skindiseases, inflammatory myopathy, insulin-dependent diabetes (type 1),insulitis, Interstitial cystitis, interstitial lung disease,interstitial lung fibrosis, iritis, ischemic re-perfusion disorder,joint inflammation, Juvenile arthritis, juvenile dermatomyositis,juvenile diabetes, juvenile onset (Type I) diabetes mellitus, includingpediatric insulin-dependent diabetes mellitus (IDDM), juvenile-onsetrheumatoid arthritis, Kawasaki syndrome, keratoconjunctivitis sicca,kypanosomiasis, Lambert-Eaton syndrome, leishmaniasis, leprosy,leucopenia, leukocyte adhesion deficiency, Leukocytoclastic vasculitis,leukopenia, lichen planus, lichen sclerosus, ligneous conjunctivitis,linear IgA dermatosis, Linear IgA disease (LAD), Loffler's syndrome,lupoid hepatitis, lupus (including nephritis, cerebritis, pediatric,non-renal, extra-renal, discoid, alopecia), Lupus (SLE), lupuserythematosus disseminatus, Lyme arthritis, Lyme disease, lymphoidinterstitial pneumonitis, malaria, male and female autoimmuneinfertility, maxillary, medium vessel vasculitis (including Kawasaki'sdisease and polyarteritis nodosa), membrano- or membranous proliferativeGN (MPGN), including Type I and Type II, and rapidly progressive GN,membranous GN (membranous nephropathy), Meniere's disease, meningitis,microscopic colitis, microscopic polyangiitis, migraine, minimal changenephropathy, Mixed connective tissue disease (MCTD), mononucleosisinfectiosa, Mooren's ulcer, Mucha-Habermann disease, multifocal motorneuropathy, multiple endocrine failure, multiple organ injury syndromesuch as those secondary to septicemia, trauma or hemorrhage, multipleorgan injury syndrome, multiple sclerosis (MS) such as spino-optical MS,multiple sclerosis, mumps, muscular disorders, myasthenia gravis such asthymoma-associated myasthenia gravis, myasthenia gravis, myocarditis,myositis, narcolepsy, necrotizing enterocolitis, and transmural colitis,and autoimmune inflammatory bowel disease, necrotizing, cutaneous, orhypersensitivity vasculitis, neonatal lupus syndrome (NLE), nephrosis,nephrotic syndrome, neurological disease, neuromyelitis optica(Devic's), neuromyelitis optica, neuromyotonia, neutropenia,non-cancerous lymphocytosis, nongranulomatous uveitis, non-malignantthymoma, ocular and orbital inflammatory disorders, ocular cicatricialpemphigoid, oophoritis, ophthalmia symphatica, opsoclonus myoclonussyndrome (OMS), opsoclonus or opsoclonus myoclonus syndrome (OMS), andsensory neuropathy, optic neuritis, orchitis granulomatosa,osteoarthritis, palindromic rheumatism, pancreatitis, pancytopenia,PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated withStreptococcus), paraneoplastic cerebellar degeneration, paraneoplasticsyndrome, paraneoplastic syndromes, including neurologic paraneoplasticsyndromes, optionally Lambert-Eaton myasthenic syndrome or Eaton-Lambertsyndrome, parasitic diseases such as Leishmania, paroxysmal nocturnalhemoglobinuria (PNH), Parry Romberg syndrome, pars planitis (peripheraluveitis), Parsonnage-Turner syndrome, parvovirus infection, pemphigoidsuch as pemphigoid bullous and skin pemphigoid, pemphigus (includingpemphigus vulgaris), pemphigus erythematosus, pemphigus foliaceus,pemphigus mucus-membrane pemphigoid, pemphigus, peptic ulcer, periodicparalysis, peripheral neuropathy, perivenous encephalomyelitis,pernicious anemia (anemia perniciosa), pernicious anemia, phacoantigenicuveitis, pneumonocirrhosis, POEMS syndrome, polyarteritis nodosa, TypeI, II, & Ill, polyarthritis chronica primaria, polychondritis (e.g.,refractory or relapsed polychondritis), polyendocrine autoimmunedisease, polyendocrine failure, polyglandular syndromes, optionallyautoimmune polyglandular syndromes (or polyglandular endocrinopathysyndromes), polymyalgia rheumatica, polymyositis,polymyositis/dermatomyositis, polyneuropathies, polyradiculitis acuta,post-cardiotomy syndrome, posterior uveitis, or autoimmune uveitis,postmyocardial infarction syndrome, postpericardiotomy syndrome,post-streptococcal nephritis, post-vaccination syndromes, preseniledementia, primary biliary cirrhosis, primary hypothyroidism, primaryidiopathic myxedema, primary lymphocytosis, which includes monoclonal Bcell lymphocytosis, optionally benign monoclonal gammopathy andmonoclonal gammopathy of undetermined significance, MGUS, primarymyxedema, primary progressive MS (PPMS), and relapsing remitting MS(RRMS), primary sclerosing cholangitis, progesterone dermatitis,progressive systemic sclerosis, proliferative arthritis, psoriasis suchas plaque psoriasis, psoriasis, psoriatic arthritis, pulmonary alveolarproteinosis, pulmonary infiltration eosinophilia, pure red cell anemiaor aplasia (PRCA), pure red cell aplasia, purulent or non purulentsinusitis, pustular psoriasis and psoriasis of the nails, pyelitis,pyoderma gangrenosum, Quervain's thyroiditis, Raynaud's phenomenon,reactive arthritis, recurrent abortion, reduction in blood pressureresponse, reflex sympathetic dystrophy, refractory sprue, Reiter'sdisease or syndrome, relapsing polychondritis, reperfusion injury ofmyocardial or other tissues, reperfusion injury, respiratory distresssyndrome, restless legs syndrome, retinal autoimmunity, retroperitonealfibrosis, Reynaud's syndrome, rheumatic diseases, rheumatic fever,rheumatism, rheumatoid arthritis, rheumatoid spondylitis, rubella virusinfection, Sampter's syndrome, sarcoidosis, schistosomiasis, Schmidtsyndrome, SCID and Epstein-Barr virus-associated diseases, sclera,scleritis, sclerodactyl, scleroderma, optionally systemic scleroderma,sclerosing cholangitis, sclerosis disseminata, sclerosis such assystemic sclerosis, sensoneural hearing loss, seronegativespondyloarthritides, Sheehan's syndrome, Shulman's syndrome, silicosis,Sjogren's syndrome, sperm & testicular autoimmunity, sphenoid sinusitis,Stevens-Johnson syndrome, stiff-man (or stiff-person) syndrome, subacutebacterial endocarditis (SBE), subacute cutaneous lupus erythematosus,sudden hearing loss, Susac's syndrome, Sydenham's chorea, sympatheticophthalmia, systemic lupus erythematosus (SLE) or systemic lupuserythematodes, cutaneous SLE, systemic necrotizing vasculitis,ANCA-associated vasculitis, optionally Churg-Strauss vasculitis orsyndrome (CSS), tabes dorsalis, Takayasu's arteritis, telangiectasia,temporal arteritis/Giant cell arteritis, thromboangiitis ubiterans,thrombocytopenia, including thrombotic thrombocytopenic purpura (TTP)and autoimmune or immune-mediated thrombocytopenia such as idiopathicthrombocytopenic purpura (ITP) including chronic or acute ITP,thrombocytopenic purpura (TTP), thyrotoxicosis, tissue injury,Tolosa-Hunt syndrome, toxic epidermal necrolysis, toxic-shock syndrome,transfusion reaction, transient hypogammaglobulinemia of infancy,transverse myelitis, traverse myelitis, tropical pulmonary eosinophilia,tuberculosis, ulcerative colitis, undifferentiated connective tissuedisease (UCTD), urticaria, optionally chronic allergic urticaria andchronic idiopathic urticaria, including chronic autoimmune urticaria,uveitis, anterior uveitis, uveoretinitis, valvulitis, vasculardysfunction, vasculitis, vertebral arthritis, vesiculobullousdermatosis, vitiligo, Wegener's granulomatosis (Granulomatosis withPolyangiitis (GPA)), Wiskott-Aldrich syndrome, or x-linked hyper IgMsyndrome.

By contrast compositions containing VISTA antagonist antibodiesaccording to the invention may be used to promote T cell immunity and totreat conditions where this is therapeutically desirable such as cancerand infectious conditions such as viral, bacterial, yeast, fungal,protozoal and parasite infections. These compositions will comprise anamount of an antagonist according to the invention effective to promoteT cell activation or proliferation or cytokine expression or othereffects of VISTA in a subject in need thereof. Such cancer conditionsinclude for example blood cancers, and solid tumors such as leukemias,lymphomas, myelodysplastic syndrome, myeloma, lung cancer, and othercancers identified herein.

It should be understood that the disease conditions identified hereinare intended to be exemplary and not exhaustive.

The subject agonists and antagonists may be combined with othertherapeutics which may be administered in the same or differentcompositions, at the same or different time. For example, the subjectagonists may be administered in a therapeutic regimen that includes theadministration of a PD-1 or PD-L1 agonist, CTLA4-Ig, a cytokine, acytokine agonist or antagonist, or another receptor agonist orantagonist.

Down Regulation of Immune Responses

Upregulating or enhancing the inhibitory function of a VISTA polypeptidemay be used to downregulate immune responses. Downregulation can be inthe form of inhibiting or blocking an immune response already inprogress, or may involve preventing the induction of an immune response.The functions of activated immune cells can be inhibited by downregulating immune cell responses or by inducing specific anergy inimmune cells, or both. For example, VISTA agonist antibodies may bind tothe VISTA polypeptide which is expressed on various immune cells andthereby down modulate the immune response. This agonist antibody may bemonospecific or multispecific, e.g., it may comprise a bispecificantibody such as a BiTE. For example, such an antibody can comprise aVISTA antigen binding moiety and another antigen binding moiety, e.g.,which targets a cell surface receptor on an immune cell, e.g., a T cell,a B cell, or a myeloid cell. Such an antibody, in addition to comprisinga VISTA antigen binding site, may comprise a binding site which binds toa B cell antigen receptor, a T cell antigen receptor, or an Fc or otherreceptor, in order to target the molecule to a specific cell population.Selection of this second antigen for the bispecific antibody providesflexibility in selection of cell population to be targeted. VISTAagonist antibodies that promote or mimic VISTA activity may enhance theinteraction of VISTA with its natural binding partners. As disclosedherein other human VISTA activating or agonist antibodies can beidentified by their ability to inhibit T cell activity or proliferationand/or based on their immunosuppressive effects in vitro orinflammatory, allergic or autoimmune disease models.

A number of art-recognized readouts of cell activation can be employedto measure, e.g., cell proliferation or effector function (e.g.,antibody production, cytokine production, phagocytosis) in the presenceof the activating agent. The ability of a test antibody to agonize orpromote the effects of human VISTA and thereby block this activation canbe readily determined by measuring the ability of the agent to affect adecrease in proliferation or effector function being measured.Accordingly, the ability of a test antibody to be immunosuppressive andto block immune activation can be determined by measuring cytokineproduction and/or proliferation at different concentrations of antigen.

Tolerance may be induced against specific antigens by co-administeringan antigen with a VISTA agonist antibody according to the invention. Forexample, tolerance may be induced to specific polypeptides Immuneresponses to allergens or foreign polypeptides to which an immuneresponse is undesirable can be inhibited. For example, patients thatreceive Factor VIII frequently generate antibodies against this clottingfactor. Co-administration of a VISTA agonist antibody according to theinvention that stimulates or mimics VISTA activity or interaction withits natural binding partner, with recombinant factor VIII may suppressthis undesired immune response.

A VISTA agonist antibody according to the invention may be used incombination with another agent that blocks the activity of costimulatoryreceptors on an immune cell or which agonizes the activity of anotherimmunosuppressive receptor or ligand expressed on immune cells in orderto down modulate immune responses. Exemplary molecules include: PD-1,PDL-1 agonists, soluble forms of CTLA-4, anti-B7-I antibodies, anti-B7-2antibodies, antagonistic antibodies targeting one or more of LAG-3,TIM-3, BTLA, B7-H4, B7H3, et al. and/or agonistic antibodies targetingone or more of CD40, CD137, OX40, GITR, CD27, CD28 or ICOS orcombinations thereof. These moieties can be combined in a singlecomposition or compound, e.g., a bispecific antibody containing a VISTAagonist antibody according to the invention and further comprisinganother immune agonist antibody or it may comprise a fusion polypeptidecontaining a VISTA agonist antibody according to the invention which isfused to another immunosuppressive polypeptide or other active agent.Alternatively these moieties may be ad ministered as separate ordiscrete entities (simultaneously or sequentially) in the same ordifferent compositions to downregulate immune cell mediated immuneresponses in a subject.

Examples of specific immunoinhibitory molecules that may be combinedwith VISTA agonist antibodies according to the invention includeantibodies that block a costimulatory signal (e.g., against CD28 orICOS), antibodies that activate an inhibitory signal via CTLA4, and/orantibodies against other immune cell markers (e.g., against CD40, CD40ligand, or cytokines), fusion proteins (e.g., CTLA4-Fc or PD-I-Fc), andimmunosuppressive drugs (e.g., rapamycin, cyclosporine A, or FK506).

In a further embodiment, bispecific antibodies containing VISTA agonistantibodies according to the invention are useful for targeting aspecific cell population, e.g., using a marker found only on a certaintype of cell, e.g., B lymphocytes, monocytes, dendritic cells, orLangerhans cells. Down regulating immune responses by activating VISTAactivity or VISTA-immune cell interactions (and thus stimulating thenegative signaling function of VISTA) is useful in downmodulating theimmune response, e.g., in situations of tissue, skin and organtransplantation, in graft-versus-host disease (GVHD), or allergies, orin autoimmune and inflammatory diseases such as systemic lupuserythematosus, IBD, RA, psoriasis and multiple sclerosis. For example,blockage of immune cell function results in reduced tissue destructionin tissue transplantation. Typically, in tissue transplants, rejectionof the transplant is initiated through its recognition as foreign byimmune cells, followed by an immune reaction that destroys thetransplant. The administration of a molecule which promotes the activityof VISTA or the interaction of VISTA with its natural bindingpartner(s), on immune cells alone or in conjunction with anotherdownmodulatory agent prior to or at the time of transplantation caninhibit the generation of a costimulatory signal. Moreover, promotion ofVISTA activity may also be sufficient to anergize the immune cells,thereby inducing tolerance in a subject.

To achieve sufficient immunosuppression or tolerance in some diseases orin some subjects, it may necessary to block the costimulatory functionof other molecules. For example, it may be desirable to block thefunction of B7-1 and B7-2 by administering a soluble form of acombination of peptides having an activity of each of these antigens orblocking antibodies against these antigens (separately or together in asingle composition) prior to or at the time of transplantation.Alternatively, it may be desirable to promote inhibitory activity ofVISTA and to further inhibit a costimulatory activity of B7-1 and/orB7-2.

The subject anti-human VISTA agonist antibodies are especially useful intreating autoimmune disease. Many autoimmune disorders are the result ofinappropriate activation of immune cells that are reactive againstself-tissue and which promote the production of cytokines andautoantibodies involved in the pathology of the diseases. Preventing theactivation of autoreactive immune cells may reduce or eliminate diseasesymptoms. Administration of the subject anti-human VISTA agonistantibodies that promote activity of VISTA or VISTA interaction with itsnatural binding partner(s), may induce antigen-specific tolerance ofautoreactive immune cells which could lead to long-term relief from thedisease. Additionally, co-administration of agents which blockcostimulation of immune cells by disrupting receptor-ligand interactionsof B7 molecules with costimulatory receptors may be useful in inhibitingimmune cell activation to prevent production of autoantibodies orcytokines which may be involved in the disease process.

Downregulation of an immune response via stimulation of VISTA activityor VISTA interaction with its natural binding partner(s) using thesubject anti-human VISTA agonist antibodies may also be useful intreating an autoimmune attack of autologous tissues. Thus, conditionsthat are caused or exacerbated by autoimmune attack (e.g., heartdisease, myocardial infarction or atherosclerosis) may be ameliorated orimproved by increasing VISTA activity or VISTA binding to its naturalbinding partner. It is therefore within the scope of the invention tomodulate conditions exacerbated by autoimmune attack, such as autoimmunedisorders (as well as conditions such as heart disease, myocardialinfarction, and atherosclerosis) by stimulating VISTA activity or VISTAinteraction with its counter receptor using the subject anti-human VISTAagonist antibodies.

As mentioned previously the efficacy of agonist anti-human VISTAantibodies according to the invention for preventing or alleviatingautoimmune and inflammatory disorders can be determined using a numberof well-characterized animal models of human autoimmune and inflammatorydiseases. Examples include murine experimental autoimmune encephalitis,systemic lupus erythematosus in MRL/lpr/lpr mice or NZB hybrid mice,murine autoimmune collagen arthritis, diabetes mellitus in NOD mice andBB rats, and murine experimental myasthenia gravis. See Paul ed.,Fundamental Immunology, Raven Press, New York, 1989, pages 840-856.

Inhibition of immune cell activation is further useful therapeuticallyin the treatment of allergies and allergic reactions, e.g., byinhibiting IgE production. The subject anti-human VISTA agonistantibodies which promote or mimic VISTA activity or VISTA interactionwith its natural binding partner(s) can be administered to an allergicsubject to inhibit immune cell-mediated allergic responses in thesubject. Stimulation of VISTA activity or interaction with its naturalbinding partner(s), can be accompanied by exposure to allergen inconjunction with appropriate MHC molecules. Allergic reactions can besystemic or local in nature, depending on the route of entry of theallergen and the pattern of deposition of IgE on mast cells orbasophils. Thus, immune cell-mediated allergic responses can beinhibited locally or systemically by administration of the subjectanti-human VISTA agonist antibodies.

Selection of Anti-VISTA Antibodies that Bind to the Same Epitope

In certain embodiments, an agonistic anti-VISTA antibody according tothe invention possesses desired functional properties such as modulationof immune stimulation and related functions. As shown in FIG. 4A-4JJ anddisclosed in the working examples, the epitopic specificity of a numberof anti-human VISTA agonist antibodies according to the invention hasbeen elucidated. As a number of antibodies which have been shown to bindto the same epitope have been found to be immunosuppressive it isexpected that other VISTA agonist antibodies may be identified whichbind to the same or overlapping epitope, i.e., they will interact withone or more of the amino acid residues of human VISTA polypeptide withwhich the exemplary VISTA agonist antibodies bind. Other antibodies withthe same epitopic specificity may be selected and/or those which havethe ability to cross-compete for binding to VISTA antigen with thedesired antibodies. For example, the epitopic specificity of a desiredantibody may be determined using a library of overlapping peptidescomprising the entire VISTA polypeptide, e.g., 15-mers or an overlappingpeptide library constituting a portion containing a desired epitope ofVISTA and antibodies which bind to the same peptides or one or moreresidues thereof in the library are determined to bind the same linearor conformational epitope. In the examples the epitopic specificity wasdetermined using Pepscan® methods which may be used to identify linearand conformational epitopes.

Modification of Agonist Antibodies According to the Invention

In addition or as an alternative to modifications made within theframework or CDR regions, antibodies according to at least someembodiments of the invention may be engineered to include modificationswithin the Fc region, typically to alter one or more functionalproperties of the antibody, such as serum half-life, complementfixation, Fc receptor binding, and/or antigen-dependent cellularcytotoxicity. Furthermore, an antibody according to at least someembodiments of the invention may be chemically modified (e.g., one ormore chemical moieties can be attached to the antibody) or be modifiedto alter its glycosylation, again to alter one or more functionalproperties of the antibody. Such embodiments are described furtherbelow. The numbering of residues in the Fc region is that of the EUindex of Kabat.

In one embodiment, the hinge region of CHI is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CHI is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcal protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S, andT256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half-life, the antibody can be altered withinthe CHI or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector functions of the antibody. For example, one or more amino acidsselected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and322 can be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity is altered can be, for example, an Fc receptor or the CIcomponent of complement. This approach is described in further detail inU.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered Clq binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551 by Idusogie etal.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351 by Bodmer et al.

In yet another example, the Fc region is modified to increase theaffinity of the antibody for an Fγ receptor by modifying one or moreamino acids at the following positions: 238, 239, 248, 249, 252, 254,255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285,286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309,312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337,338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430,434, 435, 437, 438 or 439. This approach is described further in PCTPublication WO 00/42072 by Presta. Moreover, the binding sites on humanIgGI for FcyRI, FcyRII, FcyRIII and FcRn have been mapped and variantswith improved binding have been described (see Shields, R. L. et al.(2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions256, 290, 298, 333, 334 and 339 are shown to improve binding to FcyRIII.Additionally, the following combination mutants are shown to improveFcyRIII binding: T256A/S298A, S298A/E333A, S298A/K224A andS298A/E333A/K334A. Furthermore, mutations such as M252Y/S254T/T256E orM428L/N434S improve binding to FcRn and increase antibody circulationhalf-life (see Chan C A and Carter P J (2010) Nature Rev Immunol10:301-316).

In still another embodiment, the antibody can be modified to abrogate invivo Fab arm exchange. Specifically, this process involves the exchangeof IgG4 half-molecules (one heavy chain plus one light chain) betweenother IgG4 antibodies that effectively results in b specific antibodieswhich are functionally monovalent. Mutations to the hinge region andconstant domains of the heavy chain can abrogate this exchange (seeAalberse, R C, Schuurman J., 2002, Immunology 105:9-19).

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycosylated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglyclosylationmay increase the affinity of the antibody for antigen. Such an approachis described in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861by Co et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies according to at least some embodiments of theinvention to thereby produce an antibody with altered glycosylation. Forexample, the cell lines Ms704, Ms705, and Ms709 lack thefucosyltransferase gene, FUT8 (a (1,6) fucosyltransferase), such thatantibodies expressed in the Ms704, Ms705, and Ms709 cell lines lackfucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8 celllines are created by the targeted disruption of the FUT8 gene inCHO/DG44 cells using two replacement vectors (see U.S. PatentPublication No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al.(2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 byHanai et al. describes a cell line with a functionally disrupted FUT8gene, which encodes a fucosyl transferase, such that antibodiesexpressed in such a cell line exhibit hypofucosylation by reducing oreliminating the a 1,6 bond-related enzyme. Hanai et al. also describecell lines which have a low enzyme activity for adding fucose to theN-acetylglucosamine that binds to the Fc region of the antibody or doesnot have the enzyme activity, for example the rat myeloma cell lineYB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 by Presta describesa variant CHO cell line, LecI3 cells, with reduced ability to attachfucose to Asn(297)-linked carbohydrates, also resulting inhypofucosylation of antibodies expressed in that host cell (see alsoShields, R. L. et al. (2002). Biol. Chem. 277:26733-26740). PCTPublication WO 99/54342 by Umana et al. describes cell lines engineeredto express glycoprotein-modifying glycosyl transferases (e.g.,P(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al. (1999) Nat. Biotech. 17: 176-180).Alternatively, the fucose residues of the antibody may be cleaved offusing a fucosidase enzyme. For example, the fucosidase a-L-fucosidaseremoves fucosyl residues from antibodies (Tarentino, A. L. et al. (1975)Biochem. 14:5516-23).

Another modification of the antibodies herein that is contemplated bythe invention is pegylation or the addition of other water solublemoieties, typically polymers, e.g., in order to enhance half-life. Anantibody can be pegylated to, for example, increase the biological(e.g., serum) half-life of the antibody. To pegylate an antibody, theantibody, or fragment thereof, typically is reacted with polyethyleneglycol (PEG), such as a reactive ester or aldehyde derivative of PEG,under conditions in which one or more PEG groups become attached to theantibody or antibody fragment. Preferably, the pegylation is carried outvia an acylation reaction or an alkylation reaction with a reactive PEGmolecule (or an analogous reactive water-soluble polymer). As usedherein, the term “polyethylene glycol” is intended to encompass any ofthe forms of PEG that have been used to derivatize other proteins, suchas mono (Ci-Cio) alkoxy- or aryloxy-polyethylene glycol or polyethyleneglycol-maleimide. In certain embodiments, the antibody to be pegylatedis an aglycosylated antibody. Methods for pegylating proteins are knownin the art and can be applied to the antibodies according to at leastsome embodiments of the invention. See for example, EP 0 154 316 byNishimura et al. and EP 0 401 384 by Ishikawa et al.

Methods of Engineering Antibodies

In certain embodiments, an agonist anti-VISTA antibody according to theinvention having V_(H) and V_(L) sequences can be used to create newanti-VISTA antibodies, respectively, by modifying the V_(H) and/or V_(L)sequences, or the constant regions attached thereto. Thus, in anotheraspect according to at least some embodiments of the invention, thestructural features of an anti-VISTA antibody according to at least someembodiments of the invention, are used to create structurally relatedanti-VISTA antibodies that retain at least one functional property ofthe antibodies according to at least some embodiments of the invention,such as binding to human VISTA. For example, one or more CDR regions ofone VISTA antibody or mutations thereof can be combined recombinantlywith known framework regions and/or other CDRs to create additional,recombinantly-engineered, anti-VISTA antibodies according to at leastsome embodiments of the invention, as discussed above. Other types ofmodifications include those described in the previous section. Thestarting material for the engineering method is one or more of the V_(H)and/or V_(L) sequences provided herein, or one or more CDR regionsthereof. To create the engineered antibody, it is not necessary toactually prepare (i.e., express as a protein) an antibody having one ormore of the V_(H) and/or VL sequences provided herein, or one or moreCDR regions thereof. Rather, the information contained in the sequencesis used as the starting material to create a “second generation”sequences derived from the original sequences and then the “secondgeneration” sequences is prepared and expressed as a protein.

Standard molecular biology techniques can be used to prepare and expressaltered antibody sequence. Preferably, the anti-VISTA antibody encodedby the altered antibody sequences is one that retains one, some or allof the functional properties of the anti-VISTA antibodies, respectively,produced by methods and with sequences provided herein, which functionalproperties include binding to VISTA antigen with a specific K_(D) levelor less and/or modulating immune responses and/or selectively binding todesired target cells such as for example, that express VISTA antigen.

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein. Incertain embodiments of the methods of engineering antibodies accordingto at least some embodiments of the invention, mutations can beintroduced randomly or selectively along all or part of an anti-VISTAantibody coding sequence and the resulting modified anti-VISTAantibodies can be screened for binding activity and/or other desiredfunctional properties.

Mutational methods have been described in the art. For example, PCTPublication WO 02/092780 by Short describes methods for creating andscreening antibody mutations using saturation mutagenesis, syntheticligation assembly, or a combination thereof. Alternatively, PCTPublication WO 03/074679 by Lazar et al. describes methods of usingcomputational screening methods to optimize physiochemical properties ofantibodies.

Nucleic Acid Molecules Encoding Antibodies

The invention further provides nucleic acids which encode an anti-VISTAantibody according to the invention, or a fragment or conjugate thereof.The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art. See, F. Ausubel, etal., ed. (1987) Current Protocols in Molecular Biology, GreenePublishing and Wiley Interscience, New York. A nucleic acid according toat least some embodiments of the invention can be, for example, DNA orRNA and may or may not contain intronic sequences. In a preferredembodiment, the nucleic acid is a cDNA molecule.

Nucleic acids according to at least some embodiments of the inventioncan be obtained using standard molecular biology techniques. Forantibodies expressed by hybridomas (e.g., hybridomas prepared fromtransgenic mice carrying human immunoglobulin genes as described furtherbelow), cDNAs encoding the light and heavy chains of the antibody madeby the hybridoma can be obtained by standard PCR amplification or cDNAcloning techniques. For antibodies obtained from an immunoglobulin genelibrary (e.g., using phage display techniques), nucleic acid encodingthe antibody can be recovered from the library.

Once DNA fragments encoding V_(H) and V_(L) segments are obtained, theseDNA fragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a V_(L)- or V_(H)-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. As previouslydefined, “operatively linked” means that that the two DNA fragments arejoined such that the amino acid sequences encoded by the two DNAfragments remain in-frame.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CHI,CH2 and CH3). The sequences of human heavy chain constant region genesare known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgGI, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgGI, IgG2 or IgG4 constant region. For a Fab fragment heavy chaingene, the VH-encoding DNA can be operatively linked to another DNAmolecule encoding only the heavy chain C_(Hi) constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, C_(L)—The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabat,E. A., et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and DNA fragments encompassing these regionscan be obtained by standard PCR amplification. The light chain constantregion can be a kappa (κ) or lambda (λ) constant region, but mostpreferably is a K constant region.

To create a scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly4-Ser)3, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) regions joined by the flexible linker (seee.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc.Natl. Acad. Sci., USA 85:5879-5883; McCafferty et al., (1990) Nature348:552-554).

Production of Anti-VISTA Monoclonal Antibodies

Anti-VISTA monoclonal antibodies (mAbs) and antigen-binding fragmentsaccording to the present invention can be produced by a variety oftechniques, including conventional monoclonal antibody methodology e.g.,the standard somatic cell hybridization technique of Kohler and Milstein(1975) Nature 256:495. Although somatic cell hybridization proceduresare preferred, in principle, other techniques for producing monoclonalantibody can be employed e.g., viral or oncogenic transformation of Blymphocytes.

A preferred animal system for preparing hybridomas is the murine system.Hybridoma production in the mouse is a very well-established procedure.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known. Chimeric orhumanized antibodies of the present invention can be prepared based onthe sequence of a murine monoclonal antibody prepared as describedabove. DNA encoding the heavy and light chain immunoglobulins can beobtained from the murine hybridoma of interest and engineered to containnon-murine (e.g., human) immunoglobulin sequences using standardmolecular biology techniques. For example, to create a chimericantibody, the murine variable regions can be linked to human constantregions using methods known in the art (see e.g., U.S. Pat. No.4,816,567 to Cabilly et al.). To create a humanized antibody, the murineCDR regions can be inserted into a human framework using methods knownin the art (see e.g., U.S. Pat. No. 5,225,539 to Winter and U.S. Pat.Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

According to at least some embodiments of the invention, the antibodiesare human monoclonal antibodies. Such human monoclonal antibodiesdirected against VISTA can be generated using transgenic ortranschromosomic mice carrying parts of the human immune system ratherthan the mouse system. These transgenic and transchromosomic miceinclude mice referred to herein as the HuMAb Mouse™ and KM Mouse™,respectively, and are collectively referred to herein as “human Igmice.” The HuMAb Mouse™ (Medarex Inc.) contains human immunoglobulingene miniloci that encode unrearranged human heavy μ and γ and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lon berg, et al.(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgG κmonoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lon berg, N.(1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHusza r, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546). The preparationand use of the HuMab Mouse®, and the genomic modifications carried bysuch mice, is further described in Taylor, L. et al. (1992) NucleicAcids Research 20:6287-6295; Chen, J. et al. (1993) InternationalImmunology 5:647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA90:3720-3724; Choi et al. (1993) Nature Genetics 4: 117-123; Chen, J. etal. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol.152:2912-2920; Taylor, L. et al. (1994) International Immunology6:579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851, the contents of all of which are hereby specificallyincorporated by reference in their entirety. See further, U.S. Pat. Nos.5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay;U.S. Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO99/45962, all to Lon berg and Kay; and PCT Publication No. WO 01/14424to Korman et al.

In another embodiment, human antibodies according to at least someembodiments of the invention can be raised using a mouse that carrieshuman immunoglobulin sequences on transgenes and transchomosomes, suchas a mouse that carries a human heavy chain transgene and a human lightchain transchromosome. Such mice, referred to herein as “KM Mice™”, aredescribed in detail in PCT Publication WO 02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-VISTA antibodies according to at least some embodiments of theinvention. For example, an alternative transgenic system referred to asthe Xenomouse (Abgenix, Inc.) can be used; such mice are described in,for example, U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6, 150,584and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-VISTA antibodies according to at least some embodiments of theinvention. For example, mice carrying both a human heavy chaintranschromosome and a human light chain transchromosome, referred to as“TC mice” can be used; such mice are described in Tomizuka et al. (2000)Proc. Natl. Acad Sci. USA 97:722-727. Furthermore, cows carrying humanheavy and light chain transchromosomes have been described in the art(Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and can be usedto raise anti-VISTA antibodies according to at least some embodiments ofthe invention.

Human monoclonal antibodies according to at least some embodiments ofthe invention can also be prepared using phage display methods forscreening libraries of human immunoglobulin genes. Such phage displaymethods for isolating human antibodies are established in the art. Seefor example: U.S. Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 toLadner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al.;U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S.Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and6,593,081 to Griffiths et al.

Human monoclonal antibodies according to at least some embodiments ofthe invention can also be prepared using SCID mice into which humanimmune cells have been reconstituted such that a human antibody responsecan be generated upon immunization. Such mice are described in, forexample, U.S. Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.

Immunization of Human Ig Mice

In some embodiments human Ig mice are used to raise human anti-VISTAantibodies according to the invention, e.g., by immunizing such micewith a purified or enriched preparation of VISTA antigen and/orrecombinant VISTA, or VISTA fusion protein, as described by Lon berg, N.et al. (1994) Nature 368(6474):856-859; Fishwild, D. et al. (1996)Nature Biotechnology 14: 845-851; and PCT Publication WO 98/24884 and WO01/14424. Preferably, the mice will be 6-16 weeks of age upon the firstinfusion. For example, a purified or recombinant preparation (doseranging from 0.5-500 μg) of VISTA antigen can be used to immunize thehuman Ig mice intraperitoneally.

In general transgenic mice respond when initially immunizedintraperitoneally (IP) with antigen in complete Freund's adjuvant,followed by every other week IP immunizations (up to a total of 6) withantigen in incomplete Freund's adjuvant. However, adjuvants other thanFreund's are also found to be effective. In addition, whole cells in theabsence of adjuvant are found to be highly immunogenic. The immuneresponse can be monitored over the course of the immunization protocolwith plasma samples being obtained by retroorbital bleeds. The plasmacan be screened by ELISA (as described below), and mice with sufficienttiters of anti-VISTA human immunoglobulin can be used for fusions. Micecan be boosted intravenously with antigen 3 days before sacrifice andremoval of the spleen. It is expected that 2-3 fusions for eachimmunization may need to be performed. Between 6 and 24 mice aretypically immunized for each antigen. Usually both HCo7 and HCoI2strains are used. In addition, both HCo7 and HCoI2 transgene can be bredtogether into a single mouse having two different human heavy chaintransgenes (HCo7/HCo12). Alternatively or additionally, the KM Mouse™strain can be used. In an exemplary embodiment these mice will beengineered to selectively produce human IgG2 antibodies.

Generation of Hybridomas Producing Human Monoclonal Antibodies

In certain embodiments, hybridomas producing a human monoclonalanti-VISTA antibody according to the invention may be generated usingsplenocytes and/or lymph node cells from immunized mice can be isolatedand fused to an appropriate immortalized cell line, such as a mousemyeloma cell line. The resulting hybridomas can be screened for theproduction of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toone-sixth the numbers of P3X63-Ag8.653 nonsecreting mouse myeloma cells(ATCC, CRL 1580) with 50% PEG. Cells are plated at approximately 2×105in flat bottom microtiter plate, followed by a two week incubation inselective medium containing 20% fetal Clone Serum, 18% “653” conditionedmedia, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mMHEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/mlstreptomycin, 50 mg/ml gentamycin and IX HAT (Sigma; the HAT is added 24hours after the fusion). After approximately two weeks, cells can becultured in medium in which the HAT is replaced with HT. Individualwells can then be screened by ELISA for human monoclonal IgM and IgGantibodies. Once extensive hybridoma growth occurs, medium can beobserved usually after 10-14 days. The antibody secreting hybridomas canbe replated, screened again, and if still positive for human IgG, themonoclonal antibodies can be subcloned at least twice by limitingdilution. The stable subclones can then be cultured in vitro to generatesmall amounts of antibody in tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-Sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD280using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

Generation of Transfectomas Producing Monoclonal Antibodies

In certain embodiments, an anti-VISTA antibody according to theinvention can be produced in a host cell transfectoma using, forexample, a combination of recombinant DNA techniques and genetransfection methods as is well known in the art (e.g., Morrison, s.(1985) Science 229: 1202). For example, to express the antibodies, orantibody fragments thereof, DNAs encoding partial or full-length lightand heavy chains, can be obtained by standard molecular biologytechniques (e.g., PCR amplification or cDNA cloning using a hybridomathat expresses the antibody of interest) and the DNAs can be insertedinto expression vectors such that the genes are operatively linked totranscriptional and translational control sequences. In this context,the term “operatively linked” is intended to mea n that an antibody geneis ligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype by inserting theminto expression vectors already encoding heavy chain constant and lightchain constant regions of the desired isotype such that the V_(H)segment is operatively linked to the C_(H) segments within the vectorand the V_(L) segment is operatively linked to the C_(L) segment withinthe vector. Additionally or alternatively, the recombinant expressionvector can encode a signal peptide that facilitates secretion of theantibody chain from a host cell. The antibody chain gene can be clonedinto the vector such that the signal peptide is linked in-frame to theamino terminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

Characterization of Antibody Binding to Antigen

In certain embodiments, the binding specificity of an agonisticanti-VISTA antibody according to the invention is determined by knownantibody binding assay techniques such as ELISA. In an exemplary ELISA,microtiter plates are coated with a purified antigen, herein VISTA at0.25 μg/ml in PBS, and then blocked with 5% bovine serum albumin in PBS.Dilutions of antibody (e.g., dilutions of plasma from-immunized mice)are added to each well and incubated for 1-2 hours at 37° C. The platesare washed with PBS/Tween and then incubated with secondary reagent(e.g., for human antibodies, a goat-anti-human IgG Fc-specificpolyclonal reagent) conjugated to alkaline phosphatase for 1 hour at 37°C. After washing, the plates are developed with pNPP substrate (1mg/ml), and analyzed at OD of 405-650. Preferably, mice which developthe highest titers will be used for fusions.

An ELISA assay as described above can also be used to screen forhybridomas that show positive reactivity with VISTA immunogen.Hybridomas that bind with high avidity to VISTA are subcloned andfurther characterized. One clone from each hybridoma, which retains thereactivity of the parent cells (by ELISA), can be chosen for making a5-10 vial cell bank stored at −140° C., and for antibody purification.

To purify anti-VISTA antibodies, selected hybridomas can be grown intwo-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-Sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD280using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

To determine if the selected anti-VISTA monoclonal antibodies bind tounique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (Pierce, Rockford, Ill.). Competition studies usingunlabeled monoclonal antibodies and biotinylated monoclonal antibodiescan be performed using VISTA coated-ELISA plates as described above.Biotinylated mAb binding can be detected with a strep-avidin-alkalinephosphatase probe.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed using reagents specific for antibodies of a particularisotype, e.g., IgG2's. For example, to determine the isotype of a humanmonoclonal antibody, wells of microtiter plates can be coated with{circumflex over ( )}g/ml of anti-human immunoglobulin overnight at 4°C. After blocking with 1% BSA, the plates are reacted with 1 mug/ml orless of test monoclonal antibodies or purified isotype controls, atambient temperature for one to two hours. The wells can then be reactedwith either human IgGI or human IgM-specific alkalinephosphatase-conjugated probes. Plates are developed and analyzed asdescribed above.

Anti-VISTA human IgGs can be further tested for reactivity with VISTAantigen, respectively, by Western blotting. Briefly, VISTA antigen canbe prepared and subjected to sodium dodecyl sulfate polyacrylamide gelelectrophoresis. After electrophoresis, the separated antigens aretransferred to nitrocellulose membranes, blocked with 10% fetal calfserum, and probed with the monoclonal antibodies to be tested. Human IgGbinding can be detected using anti-human IgG alkaline phosphatase anddeveloped with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis,Mo.).

In another aspect, the present invention features antibody-drugconjugates (ADCs), consisting of an antibody (or antibody fragment suchas a single-chain variable fragment (scFv) linked to a payload drug(often cytotoxic). The antibody causes the ADC to bind to the targetcancer cells. Often the ADC is then internalized by the cell and thedrug is released into the cell. Because of the targeting, the sideeffects are lower and give a wider therapeutic window. Hydrophiliclinkers (e.g., PEG4Mal) help prevent the drug being pumped out ofresistant cancer cells through MDR (multiple drug resistance)transporters.

In another aspect, the present invention features immunoconjugatescomprising an anti-VISTA antibody, or a fragment thereof, conjugated toa therapeutic agent, such as a cytotoxin, a drug (e.g., animmunosuppressant) or a radiotoxin. Such conjugates are referred toherein as “immunoconjugates”. Immunoconjugates that include one or morecytotoxins are referred to as “immunotoxins.” A cytotoxin or cytotoxicagent includes any agent that is detrimental to (e.g., kills) cells.Examples include Taxol, cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, teniposide, vincristine, vin blastine,colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents alsoinclude, for example, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thiotepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (ii) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthra mycin (AMC)), and anti-mitotic agents(e.g., vincristine and vin blastine).

Other examples of therapeutic cytotoxins that can be conjugated to anantibody according to at least some embodiments of the invention includeduocarmycins, calicheamicin, maytansines and auristatins, andderivatives thereof. An example of a calicheamicin antibody conjugate iscommercially available (Mylotarg™ Wyeth).

Cytotoxins can be conjugated to antibodies according to at least someembodiments of the invention using linker technology available in theart. Examples of linker types that have been used to conjugate acytotoxin to an antibody include, but are not limited to, hydrazones,thioethers, esters, disulfides and peptide-containing linkers. A linkercan be chosen that is, for example, susceptible to cleavage by low pHwithin the lysosomal compartment or susceptible to cleavage byproteases, such as proteases preferentially expressed in tumor tissuesuch as cathepsins (e.g., cathepsins B, C, D). For further discussion oftypes of cytotoxins, linkers and methods for conjugating therapeuticagents to antibodies, see also Saito, G. et al. (2003) Adv. Drug Deliv.Rev. 55: 199-215; Trail, P. A. et al. (2003) Cancer Immunol. Immunother.52:328-337; Payne, G. (2003) Cancer Cell 3:207-212; Allen, T. M. (2002)Nat. Rev. Cancer 2:750-763; Pastan, I. and Kreitman, R. J. (2002) Curr.Opin. Investig. Drugs 3: 1089-1091; Senter, P. D. and Springer, C. J.(2001) Adv. Drug Deliv. Rev. 53:247-264.

Antibodies of the present invention also can be conjugated to aradioactive isotope to generate cytotoxic radiopharmaceuticals, alsoreferred to as radioimmunoconjugates. Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine 131, indium 111,yttrium 90 and lutetium 177. Methods for preparing radioimmunoconjugatesare established in the art. Radioimmunoconjugates are commerciallyavailable, including Zevalin® (Biogen IDEC) and Bexxar®. (CorixaPharmaceuticals), and similar methods can be used to prepareradioimmunoconjugates using the antibodies according to at least someembodiments of the invention.

The agonist anti-human VISTA antibodies and conjugates containingaccording to at least some embodiments of the invention can be used tomodify a given biological response, and the drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, anenzymatically active toxin, or active fragment thereof, such as abrin,ricin A, Pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor or interferon-γ; or, biological response modifierssuch as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“G M-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Carriers Of Cytotoxic Agents InCancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological AndClinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985);“Analysis, Results, And Future Prospective Of The Therapeutic Use OfRadiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies ForCancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16(Academic Press 1985), and Thorpe et al., “The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62: 119-58(1982).

Bispecific Molecules

According to at least some embodiments the invention also encompassesmultispecific anti-VISTA agonist antibodies. Multispecific antibodiesare monoclonal antibodies that have binding specificities for at leasttwo different sites. In another aspect, the present invention featuresbispecific molecules comprising an anti-VISTA antibody, or a fragmentthereof, according to at least some embodiments of the invention. Anantibody according to at least some embodiments of the invention, orantigen-binding portions thereof, can be derivatized or linked toanother functional molecule, e.g., another peptide or protein (e.g.,another antibody or ligand for a receptor) to generate a bispecificmolecule that binds to at least two different binding sites or targetmolecules. The antibody according to at least some embodiments of theinvention may in fact be derivatized or linked to more than one otherfunctional molecule to generate multispecific molecules that bind tomore than two different binding sites and/or target molecules; suchmultispecific molecules are also intended to be encompassed by the term“bispecific molecule” as used herein. To create a bispecific moleculeaccording to at least some embodiments of the invention, an antibody canbe functionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other bindingmolecules, such as another antibody, antibody fragment, peptide orbinding mimetic, such that a bispecific molecule results. In certainembodiments, one of the binding specificities of the bispecificantibodies is for VISTA and the other is for any other antigen. Incertain embodiments, bispecific antibodies may bind to two differentepitopes of VISTA. Bispecific antibodies may also be used to localizecytotoxic agents to cells which express VISTA. Bispecific antibodies canbe prepared as full length antibodies or antibody fragments.

A bispecific antibody according to at least some embodiments of theinvention is an antibody which can bind simultaneously to two targetswhich are of different structure. Bispecific antibodies (bsAb) andbispecific antibody fragments (bsFab) according to at least someembodiments of the invention have at least one arm that specificallybinds to a B-cell antigen or epitope and at least one other arm thatspecifically binds a targetable conjugate.

According to at least some embodiments the invention encompasses also afusion antibody protein, which is a recombinantly producedantigen-binding molecule in which two or more different single-chainantibody or antibody fragment segments with the same or differentspecificities are linked. A variety of bispecific fusion antibodyproteins can be produced using molecular engineering. In one form, thebispecific fusion antibody protein is monovalent, consisting of, forexample, a sent with a single binding site for one antigen and a Fabfragment with a single binding site for a second antigen. In anotherform, the bispecific fusion antibody protein is divalent, consisting of,for example, an IgG with two binding sites for one antigen and two scFvwith two binding sites for a second antigen.

The invention further encompasses engineered antibodies with three ormore functional antigen-binding sites, including “Octopus antibodies”(see, e.g. US 2006/0025576A1), and “Dual Acting FAb” or “DAF” antibodiescomprising an antigen-binding site that binds to VISTA as well asanother, different antigen (see e.g. US 2008/0069820). Accordingly, thepresent invention includes bispecific molecules comprising at least onefirst binding specificity for VISTA and a second binding specificity fora second target epitope. According to at least some embodiments of theinvention, the second target epitope is an Fc receptor, e.g., humanFcyRI (CD64) or a human FcaR receptor (CD89). Therefore, the inventionincludes bispecific molecules capable of binding both to FcyR, FcaR orFcsR expressing effector cells (e.g., monocytes, macrophages orpolymorphonuclear cells (PMNs)), and to target cells expressing VISTA,respectively. These bispecific molecules target VISTA expressing cellsto effector cell and trigger Fc receptor-mediated effector cellactivities, such as phagocytosis of an VISTA expressing cells, antibodydependent cell-mediated cytotoxicity (ADCC), cytokine release, orgeneration of superoxide anion.

According to at least some embodiments of the invention in which thebispecific molecule is multispecific, the molecule can further include athird binding specificity, in addition to an anti-Fc bindingspecificity. In one embodiment, the third binding specificity is ananti-enhancement factor (EF) portion, e.g., a molecule which binds to asurface protein involved in cytotoxic activity and thereby increases theimmune response against the target cell.

The “anti-enhancement factor portion” can be an antibody, functionalantibody fragment or a ligand that binds to a given molecule, e.g., anantigen or a receptor, and thereby results in an enhancement of theeffect of the binding determinants for the Fc receptor or target cellantigen. The “anti-enhancement factor portion” can bind an Fc receptoror a target cell antigen. Alternatively, the anti-enhancement factorportion can bind to an entity that is different from the entity to whichthe first and second binding specificities bind. For example, theanti-enhancement factor portion can bind a cytotoxic T-cell (e.g., viaCD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell that resultsin an increased immune response against the target cell).

According to at least some embodiments of the invention, the bispecificmolecules comprise as a binding specificity at least one antibody, or anantibody fragment thereof, including, e.g., an Fab, Fab′, F(ab′)2, Fv,or a single chain Fv. The antibody may also be a light chain or heavychain dimer, or any minimal fragment thereof such as a Fv or a singlechain construct as described in Ladner et al. U.S. Pat. No. 4,946,778,the contents of which are expressly incorporated by reference.

In one embodiment, the binding specificity for an Fey receptor isprovided by a monoclonal antibody, the binding of which is not blockedby human immunoglobulin G (IgG). As used herein, the term “IgG receptor”refers to any of the eight γ-chain genes located on chromosome 1. Thesegenes encode a total of twelve transmembrane or soluble receptorisoforms which are grouped into three Fey receptor classes: FcyRI(CD64), FcyRII (CD32), and FcyRIII (CD16). In one preferred embodiment,the Fey receptor is a human high affinity FcyRI. The human FcyRI is a 72kDa molecule, which shows high affinity for monomeric IgG. Theproduction and characterization of certain preferred anti-Fey monoclonalantibodies are described by Fanger et al. in PCT Publication WO 88/00052and in U.S. Pat. No. 4,954,617, the teachings of which are fullyincorporated by reference herein. These antibodies bind to an epitope ofFcyRI, FcyRII or FcyRIII at a site which is distinct from the Feybinding site of the receptor and, thus, their binding is not blockedsubstantially by physiological levels of IgG. Known anti-FcyRIantibodies include mAb 22, mAb 32, mAb 44, mAb 62 and mAb 197. Thehybridoma producing mAb 32 is available from the American Type CultureCollection, ATCC Accession No. HB9469. In other embodiments, theanti-Fey receptor antibody is a humanized form of monoclonal antibody 22(H22). The production and characterization of the H22 antibody isdescribed in Graziano, R. F. et al. (1995) J. Immunol. 155 (10):4996-5002 and PCT Publication WO 94/10332. The H22 antibody producingcell line is deposited at the American Type Culture Collection under thedesignation HA022CLI and has the accession no. CRL 11177.

In still other embodiments, the binding specificity for an Fc receptoris provided by an antibody that binds to a human IgA receptor, e.g., anFc-a receptor (FcaRI (CD89)), the binding of which is preferably notblocked by human immunoglobulin A (IgA). The term “IgA receptor” isintended to include the gene product of one a-gene (FcaRI) located onchromosome 19. This gene is known to encode several alternativelyspliced transmembrane isoforms of 55 to 10 kDa. FcaRI (CD89) isconstitutively expressed on monocytes/macrophages, eosinophilic andneutrophilic granulocytes, but not on non-effector cell populations.FcaRI has medium affinity (Approximately 5×10⁻⁷ M⁻¹) for both IgAI andIgA2, which is increased upon exposure to cytokines such as G-CSF orGM-CSF (Morton, H. C. et al. (1996) Critical Reviews in Immunology16:423-440). Four FcaRI-specific monoclonal antibodies, identified asA3, A59, A62 and A77, which bind FcaRI outside the IgA ligand bindingdomain, have been described (Monteiro, R. C. et al. (1992) J. Immunol.148: 1764).

While human monoclonal antibodies are preferred, other antibodies whichcan be employed in the bispecific molecules according to at least someembodiments of the invention are murine, chimeric and humanizedmonoclonal antibodies. The bispecific molecules of the present inventioncan be prepared by conjugating the constituent binding specificities,e.g., the anti-FcR and anti-VISTA binding specificities, using methodsknown in the art. For example, the binding specificity of eachbispecific molecule can be generated separately and then conjugated toone another. When the binding specificities are proteins or peptides, avariety of coupling or cross-linking agents can be used for covalentconjugation. Examples of cross-linking agents include protein A,carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyld-dithio propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-I-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160: 1686;Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Othermethods include those described in Paulus (1985) Behring Ins. Mitt. No.78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie etal. (1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents areSATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,Ill.). When the binding moieties are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, preferablyone, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)2 or ligand×Fab fusion protein. A bispecific moleculeaccording to at least some embodiments of the invention can be a singlechain molecule comprising one single chain antibody and a bindingdeterminant, or a single chain bispecific molecule comprising twobinding determinants. Bispecific molecules may comprise at least twosingle chain molecules. Methods for preparing bispecific molecules aredescribed for example in U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881,175;5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO93/08829, and Traunecker et al., EMBOJ. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S.Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); control led Fab-armexchange (see Labrijn et al., Proc. Natl. Acad. Sci. USA 110(13):5145-50(2013)); cross-linking two or more antibodies or fragments (see, e.g.,U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81 (1985));using leucine zippers to produce bispecific antibodies (see, e.g.,Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992)); using “diabody”technology for making bispecific antibody fragments (see, e.g.,Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); andusing single-chain Fv (sFv) dimers (see, e.g. Gruber et al., J.Immunol., 152:5368 (1994)); and preparing trispecific antibodies asdescribed, e.g., in Tutt et al. J. Immunol 147: 60 (1991).

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest. For example, the FcR-antibody complexes can bedetected using e.g., an enzyme-linked antibody or antibody fragmentwhich recognizes and specifically binds to the antibody-FcR complexes.Alternatively, the complexes can be detected using any of a variety ofother immunoassays. For example, the antibody can be radioactivelylabeled and used in a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986,which is incorporated by reference herein). The radioactive isotope canbe detected by such means as the use of a γ counter or a scintillationcounter or by autoradiography.

Uses of Antagonist Antibodies and Pharmaceutical Compositions ContainingCancer Immunotherapy

Unlike tumor-targeted therapies, which are aimed at inhibiting molecularpathways that are crucial for tumor growth and development, and/ordepleting tumor cells, cancer immunotherapy is aimed to stimulate thepatient's own immune system to eliminate cancer cells, providinglong-lived tumor destruction. Various approaches can be used in cancerimmunotherapy, among them are therapeutic cancer vaccines to inducetumor-specific T cell responses, and immunostimulatory antibodies (i.e.antagonists of inhibitory receptors=immune checkpoints) to removeimmunosuppressive pathways.

Clinical responses with targeted therapy or conventional anti-cancertherapies tend to be transient as cancer cells develop resistance, andtumor recurrence takes place. However, the clinical use of cancerimmunotherapy in the past few years has shown that this type of therapycan have durable clinical responses, showing dramatic impact on longterm survival. However, although responses are long term, only a smallnumber of patients respond (as opposed to conventional or targetedtherapy, where a large number of patients respond, but responses aretransient).

By the time a tumor is detected clinically, it has already evaded theimmune-defense system by acquiring immunoresistant and immunosuppressiveproperties and creating an immunosuppressive tumor microenvironmentthrough various mechanisms and a variety of immune cells. Thus, incancer immunotherapy it is becoming increasingly clear that acombination of therapies is be required for clinical efficacy.

Combination approaches are needed and expected to increase the number ofpatients benefiting from immunotherapy and expand the number and typesof cancers that are responsive, expanding the potential cancerindications for checkpoint agents well beyond the initial indicationscurrently showing efficacy of immune checkpoint blockade as monotherapy.The combination of immunomodulatory approaches is meant to maximize theoutcomes and overcome the resistance mechanisms of most tumors to asingle approach. Thus, tumors traditionally thought of asnon-immunogenic can likely become immunogenic and respond toimmunotherapy though co-ad ministration of pro-immunogenic therapiesdesigned to increase the patient's anti-tumor immune responses.Potential priming agents are detailed herein below.

The underlying scientific rationale for the dramatic increased efficacyof combination therapy claims that immune checkpoint blockade as amonotherapy will induce tumor regressions only when there ispre-existing strong anti-tumor immune response to be ‘unleashed’ whenthe pathway is blocked. According to at least some embodiments of thepresent invention, VISTA-specific antibodies, antibody fragments,conjugates and compositions comprising same, are used for treatment ofall types of cancer in cancer immunotherapy in combination therapy.

The term “treatment” as used herein, refers to both therapeutictreatment and prophylactic or preventative measures, which in thisExample relates to treatment inflammatory side effects of cancer;however, also as described below, uses of antibodies and pharmaceuticalcompositions are also provided for treatment of infectious disease,sepsis, and/or autoimmune conditions, and/or for inhibiting anundesirable immune activation that follows gene therapy. Those in needof treatment include those already with cancer as well as those in whichthe cancer is to be prevented. Hence, the mammal to be treated hereinmay have been diagnosed as having the cancer or may be predisposed orsusceptible to the cancer. As used herein the term “treating” refers topreventing, delaying the onset of, curing, reversing, attenuating,alleviating, minimizing, suppressing, halting the deleterious effects orstabilizing of discernible symptoms of the above-described cancerousdiseases, disorders or conditions. It also includes managing the canceras described above. By “manage” it is meant reducing the severity of thedisease, reducing the frequency of episodes of the disease, reducing theduration of such episodes, reducing the severity of such episodes,slowing/reducing cancer cell growth or proliferation, slowingprogression of at least one symptom, amelioration of at least onemeasurable physical parameter and the like. For example,immunostimulatory anti-VISTA antibodies should promote T cell or NK orcytokine immunity against target cells, e.g., cancer, infected orpathogen cells and thereby treat cancer or infectious diseases bydepleting the cells involved in the disease condition. Agonisticanti-VISTA antibodies should reduce T cell or NK activity and/or or thesecretion of proinflammatory cytokines which are involved in the diseasepathology of some immune disease such as autoimmune, inflammatory orallergic conditions and thereby treat or ameliorate the diseasepathology and tissue destruction that may be associated with suchconditions (e.g., joint destruction associated with rheumatoid arthritisconditions).

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human. Preferably the mammal is a human which is diagnosedwith one of the disease, disorder or conditions described hereinabove,or alternatively one who is predisposed to at least one type of cancer.

A “therapeutically effective amount” refers to an amount of agentaccording to the present invention that is effective to treat a diseaseor disorder in a mammal. The therapeutic agents of the present inventioncan be provided to the subject alone, or as part of a pharmaceuticalcomposition where they are mixed with a pharmaceutically acceptablecarrier.

An anti-VISTA antibody, a fragment, a conjugate thereof as hereindescribed and/or a pharmaceutical composition comprising same, accordingto at least some embodiments of the present invention also can beadministered in combination therapy, i.e., combined with otherpotentiating agents and/or other therapies. According to at least someembodiments, the anti-VISTA antibody could be used in combination withany of the known in the art standard of care cancer treatment (as can befound, for example, in http://www.cancer.gov/cancertopics).

For example, the combination therapy can include an anti-VISTA antibody,a fragment, a conjugate thereof and/or a pharmaceutical compositioncomprising same, combined with at least one other therapeutic or immunemodulatory agent, other compounds or immunotherapies, or immunostimulatory strategy as described herein.

Antagonistic anti-VISTA antibodies may be used in combination withagonistic antibodies targeting immune checkpoints including anti-CTLA4mAbs, such as ipilimumab, tremelimumab; anti-PD-1 such as nivolumabBMS-936558/MDX-1106/ONO-4538, AMP224, CT-011, MK-3475, anti-PDL-1antagonists such as BMS-936559/MDX-1105, MED14736, RG-7446/M PDL3280A;Anti-LAG-3 such as IMP-321), anti-TIM-3, anti-BTLA, anti-B7-H4,anti-B7-H3; Agonistic antibodies targeting immunostimulatory proteins,including anti-CD40 mAbs such as CP-870,893, lucatumumab, dacetuzumab;anti-CD137 mAbs such as BMS-663513 urelumab, PF-05082566; anti-OX40mAbs, such as anti-OX40; anti-G ITR mAbs such as TRX518; anti-CD27 mAbs,such as CDX-1127; and anti-ICOS mAbs.

Cytokines are molecular messengers that allow the cells of the immunesystem to communicate with one another to generate a coordinated,robust, but self-limited response to a target antigen. Cytokine-basedtherapies embody a direct attempt to stimulate the patient's own immunesystem to reject cancer. The growing interest over the past two decadesin harnessing the immune system to eradicate cancer has been accompaniedby heightened efforts to characterize cytokines and exploit their vastsignaling networks to develop cancer treatments. Cytokines directlystimulate immune effector cells and stromal cells at the tumor site andenhance tumor cell recognition by cytotoxic effector cells. Numerousanimal tumor model studies have demonstrated that cytokines have broadanti-tumor activity and this has been translated into a number ofcytokine-based approaches for cancer therapy (Lee and Margolin 2011,Cancers 3(4):3856-93). A number of cytokines are in preclinical orclinical development as agents potentiating anti-tumor immune responsesfor cancer immunotherapy, including among others: IL-2, IL-7, IL-12,IL-15, IL-17, IL-18 and IL-21, IL-23, IL-27, GM-CSF, IFNα (interferona), IFNβ, and IFNγ.

Antagonist anti-VISTA antibodies and pharmaceutical compositionscontaining may also be administered in conjunction with other compoundsor immunotherapies. For example, the combination therapy can include acompound of the present invention combined with at least one othertherapeutic or immune modulatory agent, or immuno stimulatory strategy,including, but not limited to, tumor vaccines, adoptive T cell therapy,Treg depletion, antibodies (e.g. bevacizumab, Erbitux), peptides,peptibodies, small molecules, chemotherapeutic agents such as cytotoxicand cytostatic agents (e.g. paclitaxel, cisplatin, vinorelbine,docetaxel, gemcitabine, temozolomide, irinotecan, 5FU, carboplatin),immunological modifiers such as interferons and interleukins, immunostimulatory antibodies, growth hormones or other cytokines, folic acid,vitamins, minerals, aromatase inhibitors, RNAi, Histone DeacetylaseInhibitors, proteasome inhibitors, and so forth.

According to at least some embodiments, immune cells, preferably T cellscan be contacted in vivo or ex vivo with the subject therapeutic agentsto modulate immune responses. The T cells contacted with the therapeuticagents can be any cell which expresses the T cell receptor, includingα/β and γ/δ T cell receptors. T-cells include all cells which expressCD3, including T-cell subsets which also express CD4 and CDS. T-cellsinclude both naive and memory cells and effector cells such as CTL.T-cells also include cells such as ThI, TeI, Th2, Th2, Th3, ThI7, Th22,Treg, and TrI cells. T-cells also include NKT-cells and similar uniqueclasses of the T-cell lineage.

Use of Agonistic Anti-Vista Antibodies and Pharmaceutical CompositionsContaining for Treatment of Autoimmune Disease

According to at least some embodiments, anti-VISTA antibodies,fragments, conjugates thereof or a pharmaceutical composition comprisingsame, as described herein, which function as VISTA stimulatingtherapeutic agents, may be used for treating an immune system relateddisease.

Optionally, the immune system related condition comprises an immunerelated condition, autoimmune diseases as recited herein, transplantrejection and graft versus host disease and/or for blocking or promotingimmune stimulation mediated by VISTA, immune related diseases as recitedherein and/or for immunotherapy (promoting or inhibiting immunestimulation).

Optionally the immune condition is selected from autoimmune disease,transplant rejection, inflammatory disease, allergic condition or graftversus host disease. Optionally the treatment is combined with anothermoiety useful for treating immune related condition.

Thus, treatment of multiple sclerosis using the agents according to atleast some embodiments of the present invention may be combined with,for example, any known therapeutic agent or method for treating multiplesclerosis, optionally as described herein.

Thus, treatment of rheumatoid arthritis or other arthritic condition,using the subject agonist antibodies may be combined with, for example,any known therapeutic agent or method for treating rheumatoid arthritis,optionally as described herein.

Thus, treatment of IBD, using the using the subject agonist antibodiesmay be combined with, for example, any known therapeutic agent or methodfor treating IBD, optionally as described herein.

Thus, treatment of psoriasis, using the subject agonist antibodies maybe combined with, for example, any known therapeutic agent or method fortreating psoriasis, optionally as described herein.

Thus, treatment of type 1 diabetes using the subject agonist antibodiesmay be combined with, for example, any known therapeutic agent or methodfor treating type 1 diabetes, optionally as described herein.

Thus, treatment of uveitis, using the subject agonist antibodies may becombined with, for example, any known therapeutic agent or method fortreating uveitis, optionally as described herein.

Thus, treatment of psoriasis using the subject agonist antibodies may becombined with, for example, any known therapeutic agent or method fortreating psoriasis, optionally as described herein.

Thus, treatment of Sjogren's syndrome, using the subject agonistantibodies may be combined with, for example, any known therapeuticagent or method for treating for Sjogren's syndrome, optionally asdescribed herein.

Thus, treatment of systemic lupus erythematosus, using the subjectagonist antibodies may be combined with, for example, any knowntherapeutic agent or method for treating for systemic lupuserythematosus, optionally as described herein.

Thus, treatment of GVHD, using the subject agonist antibodies may becombined with, for example, any known therapeutic agent or method fortreating GVHD, optionally as described herein.

Thus, treatment of chronic or acute infection and/or hepatotoxicityassociated therewith, e.g., hepatitis, using the subject agonistantibodies may be combined with, for example, any known therapeuticagent or method for treating for chronic or acute infection and/orhepatotoxicity associated therewith, optionally as described herein.

In the above-described therapies preferably a subject with one of theaforementioned or other autoimmune or inflammatory conditions will beadministered an immunoinhibitory anti-VISTA antibody disclosed herein orantigen-binding fragment according to the invention, which antibodymimics or agonizes at least one VISTA-mediated effect on immunity, e.g.,it suppresses cytotoxic T cells, or NK activity and/or the production ofproinflammatory cytokines which are involved in the disease pathology,thereby preventing or ameliorating the disease symptoms and potentiallyresulting in prolonged disease remission, e.g., because of the inductionof Tregs which elicit T cell tolerance or prolonged immunosuppression.

The therapeutic agents and/or a pharmaceutical composition comprisingsame, as recited herein, according to at least some embodiments of theinvention, may be administered as the sole active ingredient or togetherwith other drugs in immunomodulating regimens or other anti-inflammatoryagents e.g. for the treatment or prevention of alio-orxenograft acute orchronic rejection or inflammatory or autoimmune disorders, or to inducetolerance.

Use of Agonistic Anti-Vista Antibodies and Pharmaceutical CompositionsContaining for Treatment of Sepsis

According to at least some embodiments, VISTA antibodies, fragments,conjugates thereof and/or pharmaceutical compositions as describedherein, may be used for treating sepsis. Sepsis is a potentiallylife-threatening complication of an infection. Sepsis represents acomplex clinical syndrome that develops when the initial host responseagainst an infection becomes inappropriately amplified and dysregulated,becoming harmful to the host. The initial hyperinflammatory phase(‘cytokine storm’) in sepsis is followed by a state of immunosuppression(Hotchkiss et al 2013 Lancet Infect. Dis. 13:260-268). This latter phaseof impaired immunity, also referred to as ‘immunoparalysis’, ismanifested in failure to clear the primary infection, reactivation ofviruses such as HSV and cytomegalovirus, and development of new,secondary infections, often with organisms that are not particularlyvirulent to the immunocompetent patient. The vast majority of septicpatients today survive their initial hyperinflammatory insult only toend up in the intensive care unit with sepsis-induced multi-organdysfunction over the ensuing days to weeks. Sepsis-inducedimmunosuppression is increasingly recognized as the overriding immunedysfunction in these vulnerable patients. The impaired pathogenclearance after primary infection and/or susceptibility to secondaryinfections contribute to the high rates of morbidity and mortalityassociated with sepsis.

According to at least some embodiments of the present invention, thereis provided use of a combination of the therapeutic agents and/or apharmaceutical composition comprising same, as recited herein, and aknown therapeutic agent effective for treating sepsis.

According to at least some embodiments of the present invention, thereis provided use of a combination of the therapeutic agents and/or apharmaceutical composition comprising same, as recited herein, can becombined with standard of care or novel treatments for sepsis, withtherapies that block the cytokine storm in the initial hyperinflammatoryphase of sepsis, and/or with therapies that have immunostimulatoryeffect in order to overcome the sepsis-induced immunosuppression phase.

Combination with standard of care treatments for sepsis, as recommendedby the “International Guidelines for Management of Severe Sepsis andSeptic Shock” (Del linger et al 2013 Intensive Care Med 39: 165-228),some of which are described below.

-   1. Broad spectrum antibiotics having activity against all likely    pathogens (bacterial and/or fungal—treatment starts when sepsis is    diagnosed, but specific pathogen is not identified)—example    Cefotaxime (Claforan®), Ticarcillin and clavulanate (Timentin®),    Piperacillin and tazobactam (Zosyn®), Imipenem and cilastatin    (Primaxin®), Meropenem (Merrem®), Clindamycin (Cleocin),    Metronidazole (Flagyl®), Ceftriaxone (Rocephin®), Ciprofloxacin    (Cipro®), Cefepime (Maxipime®), Levofloxacin (Levaquin®), Vancomycin    or any combination of the listed drugs.-   2. Vasopressors: example Norepinephrine, Dopamine, Epinephrine,    vasopressin-   3. Steroids: example: Hydrocortisone, Dexamethasone, or    Fludrocortisone, intravenous or otherwise Inotropic therapy: example    Dobutamine for sepsis patients with myocardial dysfunction-   4. Recombinant human activated protein C (rhAPC), such as    drotrecogin alfa (activated) (Drot AA).-   5. β-blockers additionally reduce local and systemic inflammation.-   6. Metabolic interventions such as pyruvate, succinate or high dose    insulin substitutions.    Use of Anti-VISTA Antibodies and Pharmaceutical Compositions    Containing for Reducing the Undesirable Immune Activation that    Follows Gene or Cell Therapy or Transplant

As used herein the term “gene therapy” encompasses any type of genetherapy, vector-mediated gene therapy, gene transfer, virus-mediatedgene transfer and further encompasses certain cell therapies, e.g., CART and CAR NK cell therapies. According to at least some embodiments ofthe present invention, agonist VISTA antibodies, a fragment, a conjugatethereof and/or a pharmaceutical compositions as described herein, whichtarget VISTA and have inhibitory activity on immune responses, could beused as therapeutic agents for reducing the undesirable immuneactivation that follows gene or cell therapy used for treatment ofvarious genetic diseases. Without wishing to be limited by a singlehypothesis, such antibodies have VISTA-like inhibitory activity onimmune responses and/or enhance VISTA immune inhibitory activity,optionally by inhibition of pathogenic T cells and/or NK cells.

Many gene therapy products for the treatment of genetic diseases arecurrently in clinical trials. Recent studies document therapeuticsuccess for several genetic diseases using gene therapy vectors. Genetherapy strategies are characterized by 3 critical elements, the gene tobe transferred, the target tissue into which the gene will beintroduced, and the vector (gene delivery vehicle) used to facilitateentry of the gene to the target tissue. The vast majority of genetherapy clinical trials have exploited viral vectors as very efficientdelivery vehicles, including retroviruses, lentiviruses, adenoviruses,adeno-associated viruses, pseudotype viruses and herpes simplex viruses.However, the interactions between the human immune system and all thecomponents of gene therapy vectors seem to represent one of the majorlimitations to long-lasting therapeutic efficacy. Hu man studies haveshown that the likelihood of a host immune response to the viral vectoris high. Such immune responses to the virus or the transgene productitself, resulting in formation of neutralizing antibodies and/ordestruction of transduced cells by cytotoxic cells, can greatlyinterfere with therapeutic efficacy (Seregin and Amalfitano 2010 Viruses2:2013; Mingozzi and High 2013 Blood 122:23; Masat et al 2013 DiscovMed. 15:379). Therefore, developing strategies to circumvent immuneresponses and facilitate long-term expression of transgenic therapeuticproteins is one of the main challenges for the success of gene therapyin the clinic.

Factors influencing the immune response against transgenic proteinsencoded by viral vectors include route of administration, vector dose,immunogenicity of the transgenic protein, inflammatory status of thehost and capsid serotype. These factors are thought to influenceimmunogenicity by triggering innate immunity, cytokine production, APCmaturation, antigen presentation and, ultimately, priming of naive Tlymphocytes to functional effectors (Mingozzi and High 2013 Blood122:23). Therefore, the idea to dampen immune activation by interferingwith these very mechanisms has logically emerged with the aim to inducea short-term immunosuppression, avoid the early immune priming thatfollows vector administration and promote long-term tolerance.

As a strategy to inhibit the undesirable immune activation that followsgene therapy, particularly after multiple injections, immunomodulationtreatment by targeting of two non-redundant checkpoints of the immuneresponse at the time of vector delivery was tested in animal models.Studies of vector-mediated immune responses upon adenoviral vectorinstilled into the lung in mice or monkeys showed that transienttreatment with an anti-CD40L antibody lead to suppression ofadenovirus-induced immune responses; consequently, the animals could bere-administered with adenovirus vectors. Short treatment with this Abresulted in long-term effects on immune functions and prolongedinhibition of the adenovirus-specific humoral response well beyond thetime when the Ab effects were no longer significant, pointing to thetherapeutic potential in blockade of this costimulatory pathway as animmunomodulatory regimen to enable administration of gene transfervectors (Scaria et al. 1997 Gene Ther. 4: 611; Chirmule et al 2000 7.Virol. 74: 3345). Other studies showed that co-administration ofCTLA4-Ig and an anti-CD40L Ab around the time of primary vectoradministration decreased immune responses to the vector, prolonged longterm adenovirus-mediated gene expression and enabled secondaryadenovirus-mediated gene transfer even after the immunosuppressiveeffects of these agents were no longer present, indicating that it maybe possible to obtain persistence as well as secondaryadenoviral-mediated gene transfer with transient immunosuppressivetherapies (Kay et al 1997 Proc. Natl. Acad. Sci. U.S.A 94:4686). Inanother study, similar administration of CTLA4-Ig and an anti-CD40L Ababrogated the formation of neutralizing Abs against the vector, andenabled gene transfer expression, provided the treatment wasadministered during each gene transfer injection (Lorain et al 2008Molecular Therapy 16:541). Furthermore, administration of CTLA4-Ig tomice, even as single administration, resulted in suppression of immuneresponses and prolonged transgene expression at early time points(Adriouch et al 2011 Front. Microbiol. 2: 199). However, CTLA4-Ig alonewas not sufficient to permanently wipe out the immune responses againstthe transgene product. Combined treatment targeting two immunecheckpoints with CTLA4-Ig and PD-L1 or PDL-2 resulted in synergisticimprovement of transgene tolerance at later time points, by probablytargeting two non-redundant mechanisms of immunomodulation, resulting inlong term transgene persistence and expression (Adriouch et al 2011Front. Microbiol. 2: 199).

According to at least some embodiments of the present invention, thesubject agonists may be used to overcome the limitation of immuneresponses to gene therapy, could be used for reducing the undesirableimmune activation that follows gene therapy alone or with other actives.Current approaches include exclusion of patients with antibodies to thedelivery vector, administration of high vector doses, use of emptycapsids to adsorb anti-vector antibodies allowing for subsequent vectortransduction, repeated plasma exchange (plasmapheresis) cycles to adsorbimmunoglobulins and reduce the anti-vector antibody titer.

Novel approaches attempting to overcome these limitations can be dividedinto two broad categories: selective modification of the Ad vectoritself and pre-emptive immune modulation of the host (Seregin andAmalfitano 2010 Viruses 2:2013). The first category comprises severalinnovative strategies including: (1) Ad-capsid-display of specificinhibitors or ligands; (2) covalent modifications of the entire Advector capsid moiety; (3) the use of tissue specific promoters and localadministration routes; (4) the use of genome modified Ads; and (5) thedevelopment of chimeric or alternative serotype Ads.

The second category of methods includes the use of immunosuppressivedrugs or specific compounds to block important immune pathways, whichare known to be induced by viral vectors. Immunosuppressive agents havebeen tested in preclinical studies and shown efficacy in prevention oreradication of immune responses to the transfer vector and transgeneproduct. These include general immunosuppressive agents such ascyclosporine A; cyclophosphamide; FK506; glucocorticoids or steroidssuch as dexamethasone; TLR9 blockade such as the TLR9 antagonistoligonucleotide ODN-2088; TNF-α blockade with anti-TNF-a antibodies orTNFR-Ig antibody, Erk and other signaling inhibitors such as U0126. Inthe clinical setting, administration of glucocorticoids has beensuccessfully used to blunt T cell responses directed against the viralcapsid upon liver gene transfer of adenovirus-associated virus (AAV)vector expressing human factor IX transgene to severe hemophilia Bpatients (Nathwani et al 2011 N. Engl. J. Med. 365:2357).

In contrast to the previous approaches that utilize drugs that tend to“globally” and non-specifically immunosuppress the host, more selectiveimmunosuppressive approaches have been developed. These include the useof agents which provide blockade of positive co-stimulatoryinteractions, such as between CD40 and CD154, ICOS and ICOSL, CD28 andCD80 or CD86 (including CTLA4-Ig), NKG2D and NKG2D ligands, LFA-1 andICAM, LFA-3 and CD2, 4-1BB and 4-1BBL, OX40 and OX40L, GITR and GITRLand agents that stimulate negative costimulatory receptors such asCTLA-4, PD-1, BTLA, LAG-3, TIM-1, TEVI-3, KIRs, and the receptors forB7-H4 and B7-H3. Some of these have been utilized in preclinical orclinical transplantation studies (Pilat et al 2011 Sem. Immunol.23:293).

In the above-described gene or cell therapies or in treating transplantindications preferably a subject who has or is to receive cell or genetherapy or a transplanted tissue or organ will be administered animmunoinhibitory anti-VISTA antibody disclosed herein or antigen-bindingfragment according to the invention, which antibody enhances, agonizesor mimics at least one VISTA-mediated effect on immunity, e.g., itsinhibitory effect on cytotoxic T cells or NK activity and/or itsinhibitory effect on the production of proinflammatory cytokines, or itsstimulatory effect on Tregs thereby preventing or reducing host immuneresponses against the cell or gene used in therapy or an undesiredimmune response against the transplanted cells, organ or tissue.Preferably the treatment will elicit prolonged immune tolerance againstthe transplanted or infused cells, tissue or organ. In some instances,e.g., in the case of transplanted cells, tissues or organs containingimmune cells, the immunoinhibitory anti-VISTA antibody disclosed hereinor antigen-binding fragment may be contacted with the cells, tissue ororgan prior to infusion or transplant, and/or potentially immune cellsof the transplant recipient in order to tolerize the immune cells andpotentially prevent an undesired immune response or GVHD immunereaction.

Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination ofanti-human VISTA antibodies according to the invention and optionallyanother immunosuppressive or other active agent. Thus, the presentinvention features a pharmaceutical composition comprising atherapeutically effective amount of anti-human VISTA antibodiesaccording to at least some embodiments of the present invention. Inparticular the present invention features a pharmaceutical compositioncomprising a therapeutically effective [immunosuppressive] amount of atleast one agonist anti-human VISTA antibody or antibody fragmentaccording to the present invention

A pharmaceutical composition according to at least some embodiments ofthe present invention [i.e., in the case of VISTA antagonist antibodiesdisclosed herein] may be used for the treatment of cancer, wherein thecancer is non-metastatic, invasive or metastatic, and/or for treatmentof immune related disorders, autoimmunity, allergy, GVHD, inflammationor hepatotoxicity associated with infectious disorder and/or sepsis[i.e., in the case of VISTA agonist antibodies disclosed herein].“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented. Hence, the mammal to be treated herein may have beendiagnosed as having the disorder or may be predisposed or susceptible tothe disorder. “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.Preferably, the mammal is human.

The term “therapeutically effective amount” refers to an amount of agentaccording to the present invention that is effective to treat a diseaseor disorder in a mammal. The therapeutic agents of the present inventioncan be provided to the subject alone or as part of a pharmaceuticalcomposition where they are mixed with a pharmaceutically acceptablecarrier. In many instances agonist or antagonist anti-VISTA antibodiesaccording to the invention will be used in combination with otherimmunotherapeutics or other therapeutic agents useful in treating aspecific condition.

A composition is said to be a “pharmaceutically acceptable carrier” ifits administration can be tolerated by a recipient patient. As usedherein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion).

Such compositions include sterile water, buffered saline (e.g.,Tris-HCl, acetate, phosphate), pH and ionic strength and optionallyadditives such as detergents and solubilizing agents (e.g., Polysorbate20, Polysorbate 80), antioxidants (e.g., ascorbic acid, sodiummetabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) andbulking substances (e.g., lactose, mannitol). Non-aqueous solvents orvehicles may also be used as detailed below.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions according to at least someembodiments of the invention include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants. Depending on the route ofadministration, the active compound, i.e., monoclonal or polyclonalantibodies and antigen-binding fragments and conjugates containing same,and/or alternative scaffolds, that specifically bind any one of VISTAproteins, or bispecific molecule, may be coated in a material to protectthe compound from the action of acids and other natural conditions thatmay inactivate the compound. The pharmaceutical compounds according toat least some embodiments of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, s. M., et al. (1977) J. Pharm. Sci. 66: 1-19).Examples of such salts include acid addition salts and base additionsalts. Acid addition salts include those derived from nontoxic inorganicacids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic,hydriodic, phosphorous and the like, as well as from nontoxic organicacids such as aliphatic mono- and dicarboxylic acids, phenyl-substitutedalkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic andaromatic sulfonic acids and the like. Base addition salts include thosederived from alkaline earth metals, such as sodium, potassium,magnesium, calcium and the like, as well as from nontoxic organicamines, such as N,N′-dibenzylethylenediamine, N-methylglucamine,chloroprocaine, choline, diethanolamine, ethylenediamine, procaine andthe like.

A pharmaceutical composition according to at least some embodiments ofthe invention also may include a pharmaceutically acceptableanti-oxidant. Examples of pharmaceutically acceptable antioxidantsinclude: (1) water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium meta bisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, a-tocopherol, and the like; and (3) metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositionsaccording to at least some embodiments of the invention is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin. Sterile injectable solutionscan be prepared by incorporating the active compound in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

A composition of the present invention can be administered via one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for therapeutic agentsaccording to at least some embodiments of the invention includeintravascular delivery (e.g. injection or infusion), intravenous,intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, oral,enteral, rectal, pulmonary (e.g. in halation), nasal, topical (includingtransdermal, buccal and sublingual), intravesical, intravitreal,intraperitoneal, vaginal, brain delivery (e.g. intra-cerebroventricular,intracerebral, and convection enhanced diffusion), CNS delivery (e.g.intrathecal, perispinal, and intra-spinal) or parenteral (includingsubcutaneous, intramuscular, intravenous and intradermal), transmucosal(e.g., sublingual administration), administration or administration viaan implant, or other parenteral routes of administration, for example byinjection or infusion, or other delivery routes and/or forms ofadministration known in the art. The phrase “parenteral administration”as used herein means modes of ad ministration other than enteral andtopical ad ministration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular,subcapsular, subarachnoid, intraspinal, epidural and intrasternalinjection and infusion. In a specific embodiment, a protein, atherapeutic agent or a pharmaceutical composition according to at leastsome embodiments of the present invention can be administeredintraperitoneally or intravenously.

Alternatively, an VISTA specific antibody according to the invention canbe administered via a non-parenteral route, such as a topical, epidermalor mucosal route of administration, for example, intranasally, orally,vaginally, rectally, sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a control led releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be ad ministered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition according to at least some embodiments of the invention canbe administered with a needles hypodermic injection device, such as thedevices disclosed in U.S. Pat. No. 5,399,163; 5,383,851; 5,312,335;5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-knownimplants and modules useful in the present invention include: U.S. Pat.No. 4,487,603, which discloses an implantable micro-infusion pump fordispensing medication at a control led rate; U.S. Pat. No. 4,486,194,which discloses a therapeutic device for administering medicamentsthrough the skin; U.S. Pat. No. 4,447,233, which discloses a medicationinfusion pump for delivering medication at a precise infusion rate; U.S.Pat. No. 4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. These patents are incorporated herein byreference. Many other such implants, delivery systems, and modules areknown to those skilled in the art.

In certain embodiments, the anti-VISTA antibodies can be formulated toensure proper distribution in vivo. For example, the blood-brain barrier(BBB) excludes many highly hydrophilic compounds. To ensure that thetherapeutic compounds according to at least some embodiments of theinvention cross the BBB (if desired), they can be formulated, forexample, in liposomes. For methods of manufacturing liposomes, see,e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomesmay comprise one or more moieties which are selectively transported intospecific cells or organs, thus enhance targeted drug delivery (see,e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplarytargeting moieties include folate or biotin (see, e.g., U.S. Pat. No.5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem.Biophys. Res. Commun. 153: 1038); antibodies (P. G. Bloeman et al.(1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. AgentsChemother. 39: 180); surfactant protein A receptor (Briscoe et al.(1995) Am. J Physiol. 1233: 134); pI20 (Schreier et al. (1994) J. Biol.Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett.346: 123; J. J. Killion; and I. J. Fidler (1994) Immunomethods 4:273.

In yet another embodiment, immunoconjugates of the invention can be usedto target compounds (e.g., therapeutic agents, labels, cytotoxins,radiotoxins immunosuppressants, etc.) to cells which have VISTA cellsurface receptors by linking such compounds to the antibody disclosedherein. Thus, the invention also provides methods for localizing ex vivoor in vivo cells expressing VISTA (e.g., with a detectable label, suchas a radioisotope, a fluorescent compound, an enzyme, or an enzymeco-factor). Alternatively, the immunoconjugates can be used to killcells which have VISTA cell surface receptors by targeting cytotoxins orradiotoxins to VISTA antigen.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., solublepolypeptide conjugate containing the ectodomain of the VISTA antigen,antibody, immunoconjugate, alternative scaffolds, and/or bispecificmolecule, may be coated in a material to protect the compound from theaction of acids and other natural conditions that may inactivate thecompound. The pharmaceutical compounds according to at least someembodiments of the present invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, s. M., et al. (1977) J. Pharm. Sci 66: 1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition according to at least some embodiments ofthe present invention also may include a pharmaceutically acceptableanti-oxidant. Examples of pharmaceutically acceptable antioxidantsinclude: (1) water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium meta bisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, a-tocopherol, and the like; and (3) metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Examplesof suitable aqueous and nonaqueous carriers that may be employed in thepharmaceutical compositions according to at least some embodiments ofthe present invention include water, ethanol, polyols (such as glycerol,propylene glycol, polyethylene glycol, and the like), and suitablemixtures thereof, vegetable oils, such as olive oil, and injectableorganic esters, such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of coating materials, such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositionsaccording to at least some embodiments of the present invention iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate sa Its and gelatin. Sterile injectable solutionscan be prepared by incorporating the active compound in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about I percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms according to at least some embodiments of thepresent invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

For ad ministration of the VISTA antibody disclosed herein, the dosageranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg,of the host body weight. For example dosages can be 0.3 mg/kg bodyweight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplarytreatment regime entails ad ministration once per week, once every twoweeks, once every three weeks, once every four weeks, once a month, onceevery 3 months or once every three to 6 months. Preferred dosageregimens for an antibody disclosed herein according to at least someembodiments of the present invention include 1 mg/kg body weight or 3mg/kg body weight via intravenous administration, with the antibodydisclosed herein being given using one of the following dosingschedules: (i) every four weeks for six dosages, then every threemonths; (ii) every three weeks; (iii) 3 mg/kg body weight once followedby 1 mg/kg body weight every three weeks.

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously in which case thedosage of each antibody disclosed herein ad ministered falls within theranges indicated. Anti body disclosed herein is usually administered onmultiple occasions. Intervals between single dosages can be, forexample, daily, weekly, monthly, every three months or yearly. Intervalscan also be irregular as indicated by measuring blood levels of antibodyto the target antigen in the patient. In some methods, dosage isadjusted to achieve a plasma antibody concentration of about 1-1000mug/ml and in some methods about 25-300 microgram/ml.

Alternatively, therapeutic agent can be ad ministered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of thetherapeutic agent in the patient. In general, human antibodies show thelongest half-life, followed by humanized antibodies, chimericantibodies, and nonhuman antibodies. The half-life for fusion proteinsmay vary widely. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

Having described the invention the following examples are provided tofurther illustrate the invention and its inherent advantages.

EXAMPLES Example 1: Use of Assays to Screen for ImmunosuppressiveAnti-Mouse VISTA Abs

The present inventors developed various assays to screen for putativeagonistic anti-mouse VISTA antibodies. As shown in FIG. 1 in vitro andin vivo screening assays were used to identify immunosuppressiveanti-VISTA mAbs. In the experiments in FIG. 1A purified T cells wereplated on top of anti-CD3 in the presence of the indicated mAb for 72hours. Proliferation was measured by H3 incorporation. In theexperiments in FIG. 1B purified DO11.10 T cells were stimulated by ISQpulsed APCs for 6 days in the presence of the indicated antibody.Proliferation was measured through use of CTV dilution dye. In theexperiments in FIG. 1C GVHD was induced by transfer of C57BL/6 cellsinto irradiated BALB/c recipients. Mice were injected LP. with 200 μg ofantibody on day 0, 2 and 4 post transfer and survival was analyzed. Inthe experiments in FIG. 1D mice were treated with 10 mpk of theindicated antibody 3 hours prior to administration of ConA (15 mpk) andIL-2 was analyzed in plasma at 6 by Luminex.

More particularly, in the first assay, CD4⁺ T cells were isolated andincubated with AbI, Ab2 or Ab3 before being added to anti-CD3 coatedplates. After 3 days in culture, the T cells were pulsed with tritiatedthymidine, which is incorporated by proliferating cells. Notably, bothAbI and Ab2 induced a significant reduction in the proliferative rate ofthe T cells, while Ab3 had no effect (FIG. 1 ) In a similar assay wheretransgenic T cells were stimulated with antigen pulsed APCs instead, Tcell activation was measured by proliferative dye dilution. Similar tothe anti-CD3 assay, AbI suppressed antigen-specific T cell proliferationby ˜50% (FIG. 1B). These data indicate that the Ab3 mAb blocks mVISTAfunction (i.e., enhances immune responses) whereas AbI and Ab3 stimulatemVISTA function and down regulate key immune responses.

We also determined whether Ab3 and AbI could be distinguished using invivo animal models, particularly in GVHD and ConA hepatitis models. Micewith GVHD which were treated with a control antibody (Ham Ig) hadprogressive disease and needed to be euthanized by 4 weeks post graft asexpected (FIG. 1C). Ab3 treated mice were also susceptible to GVHD, andin fact most mice died prior to the control treated group, indicatingAb3 may exacerbate disease. Conversely, all of the AbI treated miceshowed no obvious symptoms of GVHD and almost all were healthy for atleast 40 days. Specifically in these experiments mice with GVHD treatedwith a control antibody (Ham Ig) had progressive disease and needed tobe euthanized by 4 weeks post graft as expected (FIG. 1C). Ab3 treatedmice were also susceptible to GVHD, and in fact most mice died prior tothe control treated group, indicating Ab3 may exacerbate disease.Conversely, all of the AbI treated mice showed no obvious symptoms ofGVHD and almost all were healthy for at least 40 days.

In the ConA model, the inventors tested whether each VISTA antibodywould impact the well-characterized T cell cytokine response to ConA.Notably AbI, but not Ab3, induced decreased plasma cytokine levels ofIL-2 (FIG. 1D). Specifically, in the ConA model, the inventors furthertested whether each VISTA antibody would impact the well-characterized Tcell cytokine response to ConA. Notably AbI, but not Ab3, induceddecreased plasma cytokine levels of IL-2 (FIG. 1D).

Accordingly these results demonstrate that both anti-VISTA mAbs (AbI andAb2) are immunosuppressive and it has also been shown that suchimmunosuppressive anti-mouse VISTA antibodies can be distinguished frominflammatory immunosuppressive anti-mouse VISTA antibodies (Ab3). Asshown in FIG. 1 AbI is efficacious (immunosuppressive) in multipleinflammatory models including GVHD, NZB/W FI lupus-likeglomerulonephritis, concanavalin A (ConA)-induced hepatitis, collagenantibody induced arthritis (CAIA), and Imiquimod induced psoriasis. Ineach of these diseases, administration of AbI during the progression ofdisease greatly diminished pathology and/or mortality. Each model listedhas a unique requirement on T cells for disease progression. GVHD andConA are both driven by ThI T cell responses.

Example 2: Identification of Anti-VISTA Abs which SuppressesAutoimmunity in Different Autoimmune Disease Models

In the experiments in FIG. 2A-F the effects of different anti-mouseVISTA Abs were again compared in different disease models. In theexperiments in FIG. 2A NZB/W FI mice were treated 3×/week with eitherAbI or Ham Ig (200 μg) starting at 25 weeks until the end of theexperiment. “X” denotes time points where the control treated group hadall been sacrificed. In the experiment in FIG. 2B mice were treated with200 μg of antibody 3 hours prior to administration of 15 mg/kg (mpk) ofConA and survival was followed for 80 hours. In the experiment in FIG.2C mice were treated sequentially with Collagen II mAb followed by LPSand arthritis was measured by measuring for paw swelling. In theexperiments AbI and Ham-Ig were administered (200 μg) 3× every otherday. In the experiment in FIG. 2D Imiquimod was applied to the ear ofmice daily. At day 14, AbI or Ham-Ig (200 μg) were administered everyother day and ear thickness was measured with calipers. In theexperiment in the same FIG. 2E-F imiquimod was applied to the backs ofmice daily. At day 9, mice were euthanized and skin was sectioned &stained for CD3 expression by IHC.

As shown in FIG. 2A-F, in each of these experimental models,administration of AbI during the progression of the particular diseasegreatly diminished pathology and/or mortality. Each model listed has aunique requirement on T cells for disease progression. GVHD and ConA areboth driven by ThI T cell responses.

Imiquimod induced psoriasis is an IL-17/23 driven disease where T cellsare recruited into the dermal layer of the skin. AbI drastically reducedthe number of CD3+ cells in the dermis (FIGS. 2E and F), but had noimpact on splenic T cell populations (data not shown), indicating thatthis anti-mouse VISTA Ab preferentially suppressed immunity at theinflammatory lesion.

NZB/W FI lupus is a multifactorial disease with contributions from Bcells, T cells and myeloid cells. In this model, therapeuticadministration of AbI reduced proteinuria levels indicating decreaseddamage to the kidneys. Finally, CAIA does not involve adaptive immunity,instead being driven by macrophages and granulocytes. Suppression byanti-VISTA in this model indicates that the antibody may also impactupon the myeloid compartment. As such, suppressive VISTA mAb appear tomediate effects on both the T cell and innate immune compartments.

Therefore, as shown in FIG. 1 and FIG. 2 both monoclonal hamsteranti-mouse VISTA Abs AbI and AB2 induced a significant reduction in theproliferative rate of the T cells, while Ab3 had no effect (FIG. 1 ). Ina similar assay where transgenic T cells were stimulated with antigenpulsed APCs, T cell activation was measured by proliferative dyedilution. Similar to the anti-CD3 assay, AbI suppressed antigen-specificT cell proliferation by 50% (FIG. 1B). These data suggest that AbI andAb2 stimulate VISTA function and thereby down regulate key immuneresponses.

Particularly, Abl, a hamster anti-mouse VISTA antibody was efficaciousin multiple inflammatory models including GVHD, NZB/W FI lupus-likeglomerulonephritis, concanavalin A (ConA)-induced hepatitis, collagenantibody induced arthritis (CAIA), and Imiquimod induced psoriasis(FIGS. 1 and 2 ). In each of these diseases, administration of AbIduring the progression of disease greatly diminished pathology and/ormortality. Each model listed has a unique requirement on T cells fordisease progression. GVHD and ConA are both driven by ThI T cellresponses. As noted above, Imiquimod induced psoriasis is an IL-17/23driven disease where T cells are recruited into the dermal layer of theskin. Therefore, suppression by AbI in this particular autoimmune modelindicates that this antibody may also be affecting the myeloidcompartment. Therefore, these immunosuppressive anti-mouse VISTA mAb'sappear to mediate effects on both the T cell and innate immunecompartments.

Example 3: Development of Human VISTA Knock-In Mice for Use in Screeningfor Agonistic Anti-Human VISTA Abs

The previous examples relate to the isolation and characterization ofagonistic anti-mouse VISTA Abs. Heretofore an agonistic anti-human VISTAAb has never been reported in the literature. This is despite the factthat very many antagonistic anti-human VISTA antibodies have beenidentified by the present Assignee and other groups. Accordingly, priorto this invention it was uncertain whether agonistic anti-human VISTAantibodies would be identified.

Such antibodies would be highly beneficial as currently there is noapproved human therapeutics that exploit the natural function of NCR'sto suppress the immune response. Although Orencia (CTLA4-Ig) iseffective, it only acts by blocking the CD28-B7 interaction and pathwayand does not work by stimulating a down regulatory pathway. Asillustrated by the potent immunosuppressive effects of 2 differentagonistic anti-VISTA mAbs as shown in the examples which follow, theengagement of this pathway may prove to be a revolution in themanagement of different human autoimmune diseases. Moreover, theimmunosuppressive impact of anti-VISTA on both adaptive and innateautoimmune effector mechanisms sets it apart from many otheranti-inflammatory agents.

With respect to the foregoing, it was hypothesized that a desirable andnecessary reagent in screening for agonistic anti-human VISTA Abs is ahuman VISTA knock-in mouse. A human VISTA knock-in mouse has beencreated by the present Assignee (“hV-KI Mouse”). These hV-KI miceexpress human VISTA in replacement of mouse VISTA. Particularly, asshown in FIG. 3 CD4⁺ T cells, CD8⁺ T cells, Tregs (CD4⁺ FoxP3⁺), andmonocytes, CDIIb⁺, Ly6C⁺, Ly6G⁻ were isolated from the lymph nodes of WTand VISTA KI mice, and stained with a VISTA antibodies against mouse orhuman protein respectively. The expression pattern of the hV-KI isidentical to what is seen in WT mice as CD4⁺ and CD8⁺ T cells,regulatory T cells and monocytes all express consistent amounts ofsurface protein between the two strains (see FIG. 3 ).

Additionally, hV-KI mice do not develop any signs of inflammatorydisease that are observed in VISTA KO mice, indicating that hVISTA isfully functional within the mouse immune system (data not shown).Accordingly, this mouse model may be used in different assays to screenfor immunosuppressive mAbs.

Example 4: Synthesis of Putative Agonistic Anti-Human VISTA Antibodies

The sequences of different anti-human VISTA antibodies is contained inFIG. 4A-4JJ. These antibodies specifically bind to human VISTA, e.g.,VSTB49-VSTB116, and possess VISTA antagonist properties, i.e., theseantibodies inhibit the suppressive effects of VISTA on immunity when inthe IgGI format, e.g., when the antibody comprises an IgGI Fc regionwhich is wild-type, i.e., unmodified.

Among the antibodies identified in FIG. 4A-4JJ is 1E8. This murineanti-human VISTA antibody comprises the variable heavy and light chainpolypeptides set forth below and was converted by the inventors into twohuman chimeric forms. The first chimeric antibody referred to herein asINX800 was obtained by the attachment of human IgG2 heavy and lightconstant region polypeptides to the 1E8 variable heavy and light chainpolypeptides. In this first chimeric antibody none of the amino acidresidues within the IgG2 constant regions were modified.

The second chimeric antibody referred to herein as INX801 was similarlyobtained by the attachment of human IgG2 heavy and light constant regionpolypeptides to the 1E8 variable heavy and light chain polypeptides. Inthis second chimeric antibody the cysteine residue at position 127within the human IgG2 kappa chain was converted into a serine. Otherwisenone of the amino acid residues within the IgG2 constant regions weremodified.

IE8 V_(H) Polypeptide (SEQ ID NO: 57)EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEVYPDSSTINYTPSLKDKFIISRDNAKNTLYLQMIKVRSEDTALYYC ARGRGDYWGQGTSVTVSSIE8 V_(L) Polypeptide (SEQ ID NO: 58)DIQMTQSPASLSASVGETVTITCRASGNIHNYLSWYHQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQNFWSTPF TFGSGTKLEIKR.

Example 5: Evaluation of Putative Agonistic Anti-Human VISTA Antibodiesin ConA Animal Model

The effects of both chimeric IgG2 antibodies and control antibodies werecompared in a Concavalin A Hepatitis model. In this in vivo modeldifferent animals were predosed with 10 mg/kg of either chimeric IgG2antibody (INX800 or INX801) or with a control antibody 3 hours prior toConcavalin A ad ministration. 3 hours after antibody administration themice were then dosed with ConA at 12 mg/kg. These animals and thecontrols were then bled by cardiac puncture 6 hours after ConA dosing.All of the mice appeared fine, no obvious morbidity or mortality.

The blood was then analyzed for cytokine expression. Particularly, a32-plex was run using plasma obtained from the collected blood samplesusing conventional methods and cytokine test kit conventionally used forcytokine analysis. As shown in FIG. 5 the expression of severalproinflammatory cytokines was significantly suppressed in the animalsadministered INX800 or INX801 antibodies compared to the controlanimals. Particularly, GM-CSF, IL-2, IL-4, IL-6, IL-17 and TNF-α levelswere all significantly lower in the INX800 or INX801 treated animalscompared to the controls. [Reduced] expression of these cytokines wassubstantially identical in the INX800 or INX801 treated animals.

Also, the expression of certain chemokines (keratinocyte derivedchemokine or “KC”) and macrophage inflammatory protein 2 (MIP-2) weresubstantially increased in the INX800 or INX801 treated animals comparedto the controls. Again, the [increased] expression of these proteins wassubstantially identical in the INX800 or INX801 treated animals. Basedon these results both INX800 and INX801 appear to be potent VISTAagonists as they appear to elicit the analogous immunosuppressiveeffects that VISTA elicits ion the expression of various inflammatorycytokines.

Example 6: Evaluation of Putative Agonistic Anti-Human VISTA Antibodiesin Graft Versus Host Disease (GVHD) Animal Model

The effects of the same putative agonistic anti-human VISTA antibodies,INX800 and INX801 were also compared in a graft versus host disease(GVHD) animal model compared to untreated animals or controls treatedwith irrelevant antibody. In this animal model T cells were adoptivelytransferred into irradiated hosts and body-weight was measured as a readout of disease. Based on GVHD disease progression all of the Controlmice (8/8) had to be euthanized. The results of these animal studies areshown in FIG. 6 . As shown none of the INX800 or INX801 [0/8] treatedmice needed to be euthanized as GVHD was considerably depressed as aresult of treatment with INX800 or INX801 antibody. Based on theseresults both INX800 and INX801 appear to be potent VISTA agonists asthey appear to potently suppress GVHD immune responses.

Example 7: Effects of Putative Agonistic Anti-Human VISTA Antibodies onCD3-Driven T Cell Immune Responses

The effects of the same agonistic anti-human VISTA antibodies, INX800and INX801 were also compared as to their potential to suppressCD3-driven T cell immune response. In these experiments plates werecoated with OKT3 (2.5 μg/ηI). T cells were the preincubated withantibody for 30 minutes. The antibody treated T cells were then added tothe OKT3 coated plates and the T cells cultured on these plates for 72hours. As a readout of the possible effects of the antibodies onCD3-driven T cell immune responses T cell proliferation was determinedusing Tritium incorporation methods, a well-accepted method fordetecting T cell proliferation. As shown in FIG. 7 , T cellproliferation was considerably reduced in the cultured T cells whichwere treated with INX800 or INX801 antibodies compared to the control Tcell cultures.

Example 8: Effects of Putative Agonistic Anti-Human VISTA Antibodies onSpecific T Cell Populations and Total T Cell Numbers

Experiments were also affected in order to compare the possible effectsof the same anti-human VISTA antibodies, INX800 and INX801, on thenumbers of specific T cells as well as on the total number of T cells.These experiments were conducted in order to assess whether the observedeffects of the subject anti-human VISTA antibodies on cytokines and Tcells could have been attributable to cell depletion (a non-specificeffect) rather than the antibodies eliciting an immunosuppressive effectbased on their promoting specific VISTA-mediated immunosuppressiveeffects on immunity.

Both agonistic anti-human VISTA antibodies, INX800 and INX801, had nosignificant effect on the number of specific T cell populations, or onthe total number of T cells. Moreover, the results with both the INX800and INX801 antibodies were substantially the same. The results ofexemplary experiments are in FIG. 8 .

Based thereon, the observed agonistic effects of INX800 and INX801 donot appear to be attributable to cell depletion. Rather, both of theseantibodies appear to elicit an immunosuppressive effect on T cellactivation/proliferation, GVHD immune responses and the expression ofproinflammatory cytokines based on their promoting specificVISTA-mediated immunosuppressive effects on immunity.

Example 9: Summary of Effects of Different Agonistic Anti-Human VISTAAbs in Different Immune Models

As shown in Table 1 and 2 below the agonistic or immunosuppressiveeffects of different anti-human VISTA antibodies was evaluated havingthe sequences are in FIG. 4A-4JJ. To date 12 different chimericanti-human VISTA antibodies have been demonstrated to beimmunosuppressive. Some of the results obtained to date are summarizedin the Tables. Antibodies in Bin 1 all compete for binding to humanVISTA but do not compete for VISTA binding with antibodies in Bin 2.Conversely, the anti-human VISTA antibodies in Bin 2 all compete forbinding to human VISTA with each other but not with antibodies in Bin 1.

The antibody in Table 2 which is marked “inconclusive” eliciteddifferent effects, including immunosuppressive effects in the same assayor elicited ambiguous results for other reasons. As shown in Table 1 and2 a total of 12 anti-human VISTA antibodies have been isolated which areimmunosuppressive in MLR assays or ConA assays and/or other in vitro andin vivo assays or autoimmune, inflammatory or GVHD disease models andwhich mimic or agonize the immunosuppressive effects of human VISTA.Based on these results it is expected that other anti-human VISTAantibodies may be obtained by analogous methods including those havingthe same or different VISTA epitopic specificity.

Also, the experiments in FIG. 9 compare the effects of differentanti-human VISTA antibodies in ConA assays and on the expression ofselect proinflammatory cytokines and inflammation markers, i.e., IL-2, γinterferon and IL-12p70.

TABLE 1 (HUMAN OR HUMANIZED ANTI-HUMAN VISTA ANTIBODIES) 1st SuppressionAssay as IgG2 2nd MLR In MLR Assay Prolif and/or MLR Epitope as ConA HepProlif. mAb ID Group Origin IgG1 Kd, M Status Assay as IgG1)INX903|VSTB95 1 HFA ++ 1.26E−10 Tested + Hybr For (His)immunosuppression INX904|VSTB103 1 Phage, − 6.36E−10 Tested +/− yesoriginal For immunosuppression INX905|VSTB53 1 HFA ++ 2.64E−11 Tested ++Hybr (Fc) For immunosuppression INX908|VSTB92 1 HFA ++ 9.34E−11 **Tested++ Hybr (Fc) For immunosuppression INX900|VSTB50 2 HFA ++ 6.32E−10Tested +/− Hybr (Fc) For immunosuppression INX901|VSTB56 2 HFA +/−2.35E−11 Tested ++ yes Hybr (Fc) For immunosuppression INX902|VSTB63 2HFA +/− 8.30E−10 Tested ++ yes Hybr (Fc) For immunosuppressionINX906|VSTB54 2 HFA +/− 2.53E−11 Tested ++ Hybr (Fc) Forimmunosuppression INX907|VSTB66 2 HFA +/− 8.06E−11 Tested + yes Hybr(Fc) For immunosuppression INX909|VSTB67 1 HFA +/− 6.29E−11 To be testedHybr (Fc) INX913|VSTB85 1 HFA ++ 3.78E−11 To be tested Hybr (InterFAD)INX914|VSTB97 1 Phage, +/− 7.68E−10 To be tested original INX915|VSTB1061 Phage, +/− 1.67E−10 To be tested ILM INX916|VSTB107 1 Phage, ++8.90E−11 To be tested ILM INX917|VSTB110 1 Phage, +/− 2.02E−10 To betested ILM INX918|VSTB113 1 Phage, ++ 4.33E−11 To be tested ILMINX919|VSTB115 1 Phage, +/− 1.45E−10 To be tested yes ILM INX910|VSTB732 HFA +/− 2.26E−09 To be tested yes Hybr (His) INX911|VSTB76 2 HFA +/−1.31E−09 To be tested Hybr (His) INX912|VSTB84 2 HFA + 2.03E−09 To betested Hybr (InterFAD) VSTB100 1 Phage, +/− 1.48E−09 original VSTB101 1Phage, +/− 3.18E−09 original VSTB102 1 Phage, +/− 2.98E−09 originalVSTB104 1 Phage, + 6.75E−10 original VSTB105 1 Phage, + 1.15E−10 ILMVSTB108 1 Phage, + 4.94E−10 ILM VSTB109 1 Phage, +/− 1.02E−10 ILMVSTB111 1 Phage, ++ 1.71E−10 ILM VSTB112 1 Phage, ++ 1.56E−10 ILMVSTB114 1 Phage, ++ 1.52E−10 ILM VSTB116 1 Phage, ++ 2.13E−10 ILM VSTB491 HFA + 5.07E−10 Hybr (Fc) VSTB51 1 HFA ++ 1.04E−10 Hybr (Fc) VSTB59 1HFA + 1.06E−10 Hybr (Fc) VSTB65 1 HFA ++ 1.08E−09 Hybr (Fc) VSTB70 1 HFA+/− 2.23E−09 Hybr (His) VSTB81 1 HFA +/− 3.12E−10 Hybr (InterFAD) VSTB981 Phage, + 2.28E−09 original VSTB99 1 Phage, +/− 1.54E−09 originalVSTB60 2 HFA + 3.56E−10 Hybr (Fc) VSTB78 2 HFA ++ 1.13E−09 Hybr(InterFAD) VSTB74 4 HFA − 5.62E−10 Hybr (His)

TABLE 2 (MURINE ANTI-HUMAN VISTA ANTIBODIES) Antibody Bin Suppressive?MLR Prolif. Kd, M 1E8* 1 Yes ++ NT GG8 1 Yes ++ NT GA1 2 Inconclusive −NT *Shown to be immunosuppressive in 2 different IgG2 forms.

Example 10: Determination of Epitopes of Anti-Human VISTA Antibodies byB Cell Epitope Mapping

The epitopic specificity of some putative agonistic anti-human VISTAantibodies was determined using custom peptide arrays using fragments ofhuman VISTA, using proprietary methods [ProArray Ultra™] Essentially,the determination of peptide-antibody binding was performed byincubation of antibody samples with a ProArray Ultra™ peptidemicroarray, followed by incubation with a fluorescently labeledsecondary antibody. After several washing steps the ProArray Ultra™arrays were dried and scanned using a high-resolution fluorescencemicroarray scanning.

All peptides (listed below) are synthesized separately, and then boundto the ProArray Ultra™ slide surface using ProImmune's proprietarytechnology. This optimized process ensures that peptides are presentedon the array in such a manner as to closely mimic the properties of thecorresponding protein region, circumventing the inherent physiochemicalvariation of the free peptides themselves and making a compatible,combined peptide and protein array platform. The test analytes (peptidesand proteins) are dispensed onto the ProArray Ultra™ slide in discretespots and appropriate gal-files enable exact alignment of the resultingarray features back to the analyte deposited.

Peptide-antibody binding is determined by incubation of antibody samples(provided by the customer) with the ProArray Ultra™ slides, followed byincubation with a fluorescently labeled secondary antibody. After thefinal incubation and washing steps the microarrays are dried and scannedin a high-resolution microarray scanning system.

After scanning the fluorescently labeled ProArray Ultra™ slides, thescanner records an image which is evaluated using image analysissoftware-enabling interpretation and quantification of the levels offluorescent intensities associated with each fluorescent spot on thescanned microarray slide. The peptide microarray was based on anoverlapping peptide library synthesized from the human VISTA polypeptidesequence. Based on the sequence 15-mer microarray peptides, overlappingby 12 amino acids, were generated using ProImmune's ProArray Ultra™technology. Details of the peptides synthesized are listed in TABLE 3(below). ‘Position’ refers to the start and end amino acid within thepolypeptide sequence from which the peptide was derived. Synthesizedpeptides were immobilised onto ProArray Ultra™ slides in 24 identicalsub-arrays, each comprising test-peptides and control features insextuplicate spots. The peptides are shown in Table 3 below.

TABLE 3 ProArray Ultra ™ Peptide Details Peptide ID Position Sequence  1  1-15 FKVATPYSLY VCPEG (SEQ ID NO: 7)  2   4-18 ATPYSLYVCPEGQNV (SEQ ID NO: 8)  3   7-21 YSLYVCPEGQ NVTLT (SEQ ID NO: 9)  4  10-24YVCPEGQNV TLTCRL (SEQ ID NO: 10)  5  13-27 PEGQNVTLTC RLLGP(SEQ ID NO: II)  6  16-30 QNVTLTCRLL GPVDK (SEQ ID NO: 12)  7  19-33TLTCRLLGPV DKGHD (SEQ ID NO: 13)  8  22-36 CRLLGPVDKG HDVTF(SEQ ID NO: 14)  9  25-39 LGPVDKGHD VTFYKT (SEQ ID NO: 15) 10  28-42VDKGHDVTF YKTWYR (SEQ ID NO: 16) 11  31-45 GHDVTFYKT WYRSSR(SEQ ID NO: 17) 12  34-48 VTFYKTWYRS SRGEV (SEQ ID NO: 18) 13  37-51YKTWYRSSRG EVQTC (SEQ ID NO: 19) 14  40-54 WYRSSRGEV QTCSER(SEQ ID NO: 20) 15  43-57 SSRGEVQTCS ERRPI (SEQ ID NO: 21) 16  46-60GEVQTCSERR PIRNL (SEQ ID NO: 22) 17  49-63 QTCSERRPIR NLTFQ(SEQ ID NO: 23) 18  52-66 SERRPIRNLTF QDLH (SEQ ID NO: 24) 19  55-69RPIRNLTFQD LHLHH (SEQ ID NO: 25) 20  58-72 RNLTFQDLHL HHGGH(SEQ ID NO: 26) 21  61-75 TFQDLHLHH GGHQAA (SEQ ID NO: 27) 22  64-78DLHLHHGGH QAANTS (SEQ ID NO: 28) 23  67-81 LHHGGHQAA NTSHDL(SEQ ID NO: 29) 24  70-84 GGHQAANTS HDLAQR (SEQ ID NO: 30) 25  73-87QAANTSHDL AQRHGL (SEQ ID NO: 31) 26  76-90 NTSHDLAQR HGLESA(SEQ ID NO: 32) 27  79-93 HDLAQRHGL ESASDH (SEQ ID NO: 33) 28  82-96AQRHGLESAS DHHGN (SEQ ID NO: 34) 29  85-99 HGLESASDH HGNFSI(SEQ ID NO: 35) 30  88-102 ESASDHHGN FSITMR (SEQ ID NO: 36) 31  91-105SDHHGNFSIT MRNLT (SEQ ID NO: 37) 32  94-108 HGNFSITMR NLTLLD(SEQ ID NO: 38) 33  97-111 FSITMRNLTLL DSGL (SEQ ID NO: 39) 34 100-114TMRNLTLLDS GLYCC (SEQ ID NO: 40) 35 103-117 NLTLLDSGLY CCLVV (SEQ IDNO: 41) 36 106-120 LLDSGLYCCLV VEIR (SEQ ID NO: 42) 37 109-123SGLYCCLVVEI RHHH (SEQ ID NO: 43) 38 112-126 YCCLVVEIRH HHSEH(SEQ ID NO: 44) 39 115-129 LVVEIRHHHS EHRVH (SEQ ID NO: 45) 40 118-132EIRHHHSEHR VHGAM (SEQ ID NO: 46) 41 121-135 HHHSEHRVH GAMELQ(SEQ ID NO: 47) 42 124-138 SEHRVHGAM ELQVQT (SEQ ID NO: 48) 43 127-141RVHGAMELQ VQTGKD (SEQ ID NO: 49) 44 130-144 GAMELQVQT GKDAPS(SEQ ID NO: 50) 45 133-147 ELQVQTGKD APSNCV (SEQ ID NO: 51) 46 136-150VQTGKDAPS NCVVYP (SEQ ID NO: 52) 47 139-153 GKDAPSNCV VYPSSS(SEQ ID NO: 53) 48 142-156 APSNCVVYPS SSQDS (SEQ ID NO: 54) 49 145-159NCVVYPSSSQ DSENI (SEQ ID NO: 55) 50 148-162 VYPSSSQDSE NITAA(SEQ ID NO: 56)

The results of this epitope analysis with particular anti-human VISTAantibodies are summarized in FIG. 4A-4JJ.

Example 11: Epitope Binning Assay

Additionally the epitopic binding properties of some anti-human VISTAantibodies having sequences shown in FIG. 4A-4JJ were characterized byplacing these antibodies into different epitope “bins” based on theirbinding characteristics as described below.

Methods: ProteOn XPR36 system (BioRad) was used to perform epitopebinning. ProteOn GLC chips (BioRad, Cat #176-5011) were coated with twosets of 6 monoclonal antibodies (mAbs) using the manufacturerinstructions for amine-coupling chemistry (BioRad, cat #176-2410).Competing mAbs were pre-incubated in excess (250 nM final concentration)with human VISTA (25 nM final concentration) for 4 hours at roomtemperature and 6 at a time were run over the chip coated with thepanels of coated mAbs with an association time of 4 minutes followed bydissociation for 5 minutes. Following each run, the chips wereregenerated with 100 nM phosphoric acid.

The data analysis involved grouping all sensorgrams by ligand andapplying an alignment wizard, which automatically performs an X and Yaxis alignment, and artifact removal. An Interspot correction was thenapplied to the data.

A non-competing mAb was defined as having a binding signal the sameor >Al signal (binding to human VISTA only). A competing mAb was definedas having binding signal<<Al signal {i.e., binding to human VISTA only).For example VSTB49 and VSTB51 complexed with VISTA did not bind to theVSTB85 coated on the chip and therefore were classified as competing forthe same binding site on VISTA as VSTB85. The results of this binninganalysis with particular anti-human VISTA antibodies are summarized inFIG. 4A-4JJ.

Example 12: Epitope Mapping of Anti-VISTA Antibodies UsingHydrogen/Deuterium (HD) Exchange Studies

Antibody epitopes of anti-VISTA antibodies may be identified by variousmethods such as ala nine scanning and Hydrogen/Deuterium (HD) Exchangeand overlapping peptide arrays as described in the previous Example.Another exemplary means for identifying epitopes of putative agonisticanti-human VISTA antibodies is described below.

To identify the epitopes for VSTB50, 60, 95 and 112 on human VISTA,solution hydrogen/deuterium exchange-mass spectrometry (HDX-MS) wasperformed using the corresponding Fabs. For H/D exchange, the proceduresused to analyze the Fab perturbation were similar to that describedpreviously (Hamuro et al, J. Biomol. Techniques 14:171-182, 2003; Hornet al, Biochemistry 45:8488-8498, 2006) with some modifications. Fabswere prepared from the IgGs with papain digestion and Protein A captureusing Pierce Fab Preparation Kit (Thermo Scientific, Cat #44985). Thehuman VISTA protein sequence contains six N-linked glycosylation sites.To improve the sequence coverage, the protein was deglycosylated withPNGase F. The deglycosylated VISTA protein was incubated in a deuteratedwater solution for predetermined times resulting in deuteriumincorporation at exchangeable hydrogen atoms. The deuterated VISTAprotein was in complex with a Fab of VSTB50, VSTB60, VSTB95 or VSTB112in 46 deuterium oxide (D20) at 4° C. for 30 sec, 2 min, 10 min and 60min. The exchange reaction was quenched by low pH and the proteins weredigested with pepsin. The deuterium levels at the identified peptideswere monitored from the mass shift on LC-MS. As a reference control,VISTA protein was processed similarly except that it was not in complexwith the Fab molecules. Regions bound to the Fab were inferred to bethose sites relatively protected from exchange and, thus, containing ahigher fraction of deuterium than the reference VISTA protein. About 94%of the protein could be mapped to specific peptides.

The solution HDX-MS perturbation maps of VISTA with VSTB50/VSTB60, andVSTB95/VSTB112 were mapped and two epitope groups were identified.Anti-VISTA VSTB50 recognizes the same epitope as VSTB60 does; VSTB95binds to another epitope region as VSTB112 does on VISTA. Anti-VISTAVSTB50 and 60 share the same epitope which comprises segments,103NLTLLDSGL111 (SEQ ID NO:59), and 136VQTGKDAPSNC146 (SEQ ID NO:60)Anti-VISTA VSTB95 and VSTB112 appear to target similar epitopes,comprising segments 27PVDKGHDVTF36 (SEQ ID NO:61), and 54RRPIRDLTFQDL65(SEQ ID NO:62). These HDX-MS results provide the peptide level epitopesfor exemplary anti-VISTA antibodies having the sequences identified inFIG. 4A-4JJ. There were no overlapping epitope regions for these twoepitope groups. These results are in agreement with the previouscompetition binning data in that they do not compete with each other.Again the epitope analysis results for various anti-human VISTAantibodies analyzed as described herein is summarized in FIG. 4A-4JJ.

Example 13: Assays for Identifying Agonist Anti-Human VISTA Antibodies

As disclosed herein, we have identified a dozen agonistic anti-humanVISTA antibodies and should be in possession of others once furthercorroborative experiments in the afore-described immune models areconducted or repeated with other antibodies. The antibodies identifiedin FIG. 4A-4JJ by “VSTB” designations are fully-human, high-affinitycynomolgus monkey cross-reactive anti-VISTA antibodies (library affinityrange 298-24 pM for human and 443-26 pM for cynomolgus monkey) which,based on the successful isolation of numerous agonistic anti-humanantibodies as described herein should give rise to the identification ofother agonistic anti-human VISTA antibodies, especially others whichbind to epitope group 1 or 2. These methods are described below.

Functional Screening In Vitro

Direct CD4 Mediated:

In this approach CD4⁺ T cells are isolated by negative selection fromhV-KI splenocytes. 1×105 T cells will then be incubated with each of the50 VISTA mAb (20 μg/ml) or an isotype control for 30 minutes on ice. TheT cells and antibody will then be placed on anti-CD3 coated 96-wellflat-bottom plates and cultured for 72 hours. At the 72-hour time point,tritiated thymidine will be added to the culture for 8 hours to measureproliferation by H3 incorporation. Using this assay, we can screen all50 mAb in a single experiment in technical triplicates. Each antibodywill be tested in three independent experiments to confirm activity. MAbthat decrease proliferation to a statistically significant extent incomparison to the isotype control will be identified as “suppressive.”All suppressive mAb identified in the initial screen will then beretested in the same assay, to generate a dose-response curve. Eachantibody will be tested at half-log dilutions (30 g/ml→0.01 μg/ml) andIC50 values will be calculated for proliferation. All antibodies thatare identified as suppressive in the hV-KI assay will be confirmed onprimary human T cells, and ranked by IC50 scores for proliferation.

NHP Cross-Reactivity Assay:

In this assay we will screen for functional activity in a relevant toxspecies, Macaca fascicularis (hereafter referred to as non humanprimates or “NHPs”), through the identical CD3 mediated proliferationassays described for mouse and human, through use of the CD3 clone SP34which drives potent T cell proliferation. Whole blood from NHPs will beobtained from World Wide Primates (Florida, USA), and T cells will beisolated through magnetic separation. The T cells will be incubated withantibody and cultured on CD3 coated plates for 72 hours. Proliferationwill be measured by tritium incorporation and IC50 scores will begenerated for each antibody.

Functional Screening Using In Vivo Animal Models

1. Testing of Vista Agonist Antibodies According to the Invention inConcanavalin A-Induced Hepatitis Animal Model.

Autoimmune hepatitis (AIH) is a chronic inflammatory disease of theliver, characterized by the loss of self-tolerance leading to B and Tcell responses against the liver. The ConA model represents thebest-characterized system for understanding the pathogenesis of AIH.ConA is a lectin that binds to specific sugar moieties, which areenriched in the liver. The modification of these sugar residues by ConAresults in rapid CD4⁺ T cell activation through interaction withmodified MHC structures expressed by liver macrophages. An intense, buttransient, cytokine production occurs with most canonica IT cellcytokines (IL-2, IL-3, IFNy and TNFa) reaching peak plasma levels within4-6 hours. Notably, ConA induced inflammation can be blocked bydepleting CD4+ T cells. The ConA model with hV-KI mice may be used toconfirm suppressive activity of agonistic anti-VISTA mAbs according tothe invention. Mice are weighed and treated with 10 mpk of anti-VISTAantibody or the appropriate isotype control 3 hours prior to injectionwith 15 mpk of ConA. The anti-VISTA mAbs are ad ministered LP. whileConA is injected via the tail-vein in these mice. At the 6-hourtime-point post ConA administration, the mice are euthanized and bloodis collected. The plasma fraction is then be analyzed for plasmacytokines by a multiplex assay for 32 cytokines. Each antibody is testedtwo times in independent experiments to confirm activity. For eachcytokine in the 32-plex, a one-way ANOVA will be performed, with aDunnett's post-test to compare each anti-VISTA antibody to the isotypecontrol. The tested anti-VISTA mAb is ranked based upon efficacy ofcytokine suppression (how much was the cytokine suppressed) andvariability (how consistent is the suppression within each experimentand between experiments). Additional emphasis is placed on mAb thatsuppress cytokines that are canonically associated with T cellactivation.

As disclosed in a related PCT application filed on even date and supra,several anti-human VISTA antibodies according to the invention werescreened in the ConA model and were efficacious (immunosuppressive)therein, i.e., they suppressed ConA-induced cytokine production andpromoted survival and in particular suppressed the expression ofcytokines involved in T cell activation including IL-2. Particularly,the inventors tested INX800, INX801, and INX903 and INX904 as well asagonist anti-murine VISTA antibodies and all were efficacious(immunosuppressive) in the ConA hepatitis model. Therefore, agonistanti-human VISTA antibodies according to the invention should be usefulin treating/preventing inflammation and hepatotoxicity associated withsome chronic and acute infectious conditions such as hepatitis.

2. Testing of Vista Agonist Antibodies According to the Invention inGraft Versus Host Disease Animal Models

GVHD is a systemic disease mediated by adoptive transfer of allogeneic Tcells into an irradiated host. There are five major steps that arecritical in the pathogenesis of GVHD; 1) Damage to the host, mostcommonly in the form of the irradiation event that precedes the T celltransfer; 2) Activation of the allogeneic T cells by both host and donorAPCs; 3) Expansion of the T cells in the lymph nodes and spleen; 4)Trafficking into peripheral sites such as the skin, gut, liver and lung;and 5) Damage to the host driven by T cells and also recruited myeloidcells. In certain models, such as FI→Parental strain, a chronic GVHDoccurs that is a suitable model for lupus as the mice developanti-nuclear mAb and immune complex mediated glomerula r nephritis. Ofnote, genetic deletion of VISTA from the donor T cells results in a moreaggressive form of GHVD than seen in mice receiving WTT cells.

This assay may be used to identify and rank agonism of agonisticanti-human VISTA candidates. Also this assay may be used to confirm thatagonist antibodies according to the invention may be used to treat orprevent GVHD. In this model BALB/c mice are lethally irradiated andgiven allogeneic bone marrow and splenic T cells from hV-KI mice toinduce GVHD; with one group not receiving T cells as a negative control.Mice receiving the allogeneic T cells are split into the control Iggroup and the treatment groups. Up to four unique VISTA mAb will be usedin a single experiment, with eight mice per group, and two replicateexperiments will be conducted. 10 mpk or another dose of antibody isadministered at the time of T cell transfer, as well as at days 2 and 4post transfer. The body weight of each mouse will be tracked, and anymouse that loses more than 20% of its initial starting bodyweight willbe sacrificed. Kaplan Meier curves are generated for each experimentwith a log-rank statistical test comparing each anti-VISTA antibody tothe control. Should all four VISTA mAb fully protect against GVHD, thendose response assays will be run in the GVHD model with groups beingtreated with 10, 3, 1 and 0.3 mpk of antibody. LD50 values will becalculated for each antibody.

As disclosed in a related application filed on even date and supra anumber of agonist anti-human VISTA antibodies according to the inventionwere evaluated in this animal model. These tested antibodies all wereefficacious (immunosuppressive) in this model, i.e., they reduced thesymptoms of the disease, slowed disease progression, reduceddisease-associated weight loss and promoted survival. Particularly, eachof INX800, INX801, INX901, INX902, INX903 and INX904 were evaluated andwere demonstrated to alleviate or prevent disease symptoms in thisanimal model. Also, it was determined using the A and B forms of INX901that either the A or B form were equally effective in the GVHD animalmodel.

3. Testing of Vista Agonist Antibodies According to the Invention in anAnimal Model of Inflammatory Bowel Disease.

Inflammatory bowel diseases (IBD), Crohn's disease and ulcerativecolitis result from incompletely defined and complex interactionsbetween host immune responses, genetic susceptibility, environmentalfactors, and the enteric luminal contents. Recent genome-wideassociation studies report associations between immune cell regulatorygenes and IBD susceptibility. Both innate and adaptive immune cellintrinsic genes are represented in these studies, indicating a centralrole for these cell populations in IBD pathogenesis. There currentlyexist more than 50 animal models of human IBD. While no one modelperfectly phenocopies human IBD, many are useful for studying variousaspects of human disease, including disease onset and progression andthe wound-healing response.

In one well established IBD model intestinal inflammation is initiatedwith syngeneic splenic CD4⁺ CD45RB^(hi) T cell adoptive transfer into Tand B cell deficient recipient mice. The CD4+ CD45RBhi T cell populationcontains mainly naive T cells primed for activation that are capable ofinducing chronic small bowel and colonic inflammation. This methodallows the researcher to modify key experimental variables, includingboth innate and adaptive immune cell populations, to answer biologicallyrelevant questions relating to disease pathogenesis. Additionally, thismethod provides precise initiation of disease onset and awell-characterized experimental time course permitting the kinetic studyof clinical features of disease progression in mice. Intestinalinflammation induced by this method shares many features with human IBD,including chronic large and small bowel transmural inflammation,pathogenesis driven by cytokines such as TNF and IL-12, and systemicsymptoms such as wasting. Thus, it is an ideal model system for studyingthe pathogenesis of human IBD.

As disclosed in a related PCT application filed on even date anagonistic anti-human VISTA antibody according to the invention (INX901)was tested and shown to be efficacious in this IBD model. Particularlythis agonist antibody was demonstrated to suppress cytokine levels andto effectively prevent or inhibit (i) colitis related weight loss, (ii)weight loss associated with colitis progression, (iii) colon shortening,(iv) the recruitment of inflammatory infiltrates to the colon and (v)the development of colitis. Therefore, agonist VISTA antibodiesaccording to the invention may be used in the treatment of IBD andrelated inflammatory and intestinal conditions.

4. Testing of VISTA Agonist Antibodies According to the Invention inLupus Animal Models.

Lupus is an autoimmune or inflammatory condition with symptoms includingkidney inflammation, increased proteinuria, and splenomegaly. There are4 types of lupus of which Systemic Lupus Erythematosus or (“SLE”) is themost common form. This disease can be mild or severe and can affectmajor organ systems. Lupus is an autoimmune condition of unknown causethat may result in inflammation of the kidneys—called lupusnephritis—which can affect the body's ability to filter waste from theblood, and or if severe may result in kidney damage requiring dialysisor kidney transplant. Also SLE may result in an increase in bloodpressure in the lungs—called pulmonary hypertension—which can causedifficulty breathing. Further SLE may cause Inflammation of the nervoussystem and brain which can cause memory problems, confusion, headaches,and strokes. Further SLE may result in inflammation in the brain's bloodvessels which can cause high fevers, seizures, and behavioral changes.Also SLE may result in hardening of the arteries or coronary arterydisease—the buildup of deposits on coronary artery walls—can lead to aheart attack.

As disclosed in a related PCT application filed on even date agonisticanti-human VISTA antibodies according to the invention (INX903, INX901,INX901-A and INX901-B) and anti-murine VISTA antibodies were tested andshown to be efficacious in different lupus models including the MRL/lprlupus model, the NZBWF-1 lupus model and the B6D2F model. The B6D2Fmodel is a murine model wherein SLE is induced by the transfer of humanVISTA knock-in DDE1 CD8 depleted splenocytes (donor) into a B6D2F1 host(recipient) In this model, donor CD4 T cell polyclonal activation drivescognate host B cell activation, expansion, and their production ofautoantibodies leading to renal disease. Lupus-like features of B6 CD8depleted transferred to B6D2F1 model include: (1) Immune complexglomerulonephritis; (2) anti-nuclear abs; (3) anti-dsDNA abs; and (4)anti-RBC abs (Coombs positivity). Additionally, this model meetssex-based differences in renal disease severity.

In 3 different lupus models agonistic anti-human and murine VISTAantibodies were demonstrated to be efficacious and to reduce theincidence of lupus disease development, disease progression, reduceproteinuria levels, inhibit nephritis and kidney damage, reduce T cellactivation and accumulation, reduce B cell activation and accumulation,and to inhibit autoantibody production. Particularly, INX903, INX901,INX901-A and INX901-B were shown to (i) reduce T cell proliferation andactivation, (ii) reduce cognate B cell activation (MHCII expression) andaccumulation, reduce splenomegaly, reduce anti-dsDNA IgG auto-antibodyproduction and to reduce type I interferon signature. Also theseimmunosuppressive effects were not impacted by whether the human IgG2constant region of the antibody was in the A or B form. Therefore,agonist VISTA antibodies according to the invention may be used in thetreatment of lupus and related inflammatory and autoimmune conditions.

5. Testing of Vista Agonist Antibodies in a Psoriasis Animal Model

Imiquimod (IMQD) Induced Psoriasis Model

The ability of anti-VISTA antibodies to treat psoriasis was evaluatedusing the Imiquimod (IMQD) induced Psoriasis Model. Imiquimod (IMQD) isa commercially available cream containing TLR7/8 agonists that is widelyused for dermatological conditions such as viral infections andmelanoma. Application of IMQD to the skin over multiple days results inthickening of the epidermis via proliferation of the keratinocytes.Additionally, an immunological infiltration into the dermis layeroccurs, with populations of both T cells and myeloid cells. Recurrentadministration of IMQD creates a skin lesion similar to what is observedin patients with Psoriasis. IL-17 and IL-23 are thought to be the majorcytokines involved in the immune response to IMQD.

As disclosed in a related PCT application filed on even date anagonistic anti-VISTA antibody was tested and shown to be efficacious inthis psoriasis model. Particularly, this antibody reduced the number ofCD3⁺ T cells infiltrating Imiquimod treated skin. Based on the observedresults VISTA agonist antibodies may be used in the treatment orprevention of psoriasis and other T cell mediated autoimmune orinflammatory skin conditions.

6. Testing of VISTA Agonist Antibodies in Arthritis Animal Model

The immunosuppressive effects of anti-VISTA antibodies to treatarthritis may be tested in different animal models. As disclosed in arelated PCT application filed on even date agonistic anti-murine andanti-human VISTA antibodies were tested and shown to be efficacious in awell-accepted arthritis model, i.e., the Collagen induced arthritis orCIA Model. INX800, INX901, INX902 and INX903 as well as a hamsteranti-murine anti-VISTA antibodies were all tested in this arthritismodel. Disease development was assessed by measuring inflammationswelling in the affected joints over time. Clinical scoring wasaccomplished by awarding a score of 1 for each swollen digit, a score of5 for a swollen footpad and a score of 5 for a swollen wrist or ankle(Charles River Labs scoring system), which added together give a maximalscore of 60 for each animal.

As shown in this PCT application each of these antibodies decreased thearthritis disease and INX901 and INX902 significantly decreased diseasescope. Based on these results anti-human VISTA agonist antibodies may beused in the treatment or prevention of rheumatoid arthritis and otherinflammatory or autoimmune conditions.

Example 14: Evaluation of the Role of the Human IgG2 Backbone on a-HumanVISTA Antibody INX901 Agonist/Immune-Suppressive Activity in DifferentIn Vitro and In Vivo Models

Antibodies on a native human IgG2 backbone exist as a mixture ofisoforms caused by disulfide bond shuffling among cysteines present inthe heavy chain hinge, CHI, and light chain (Zhang, A., (2015),“Conformational difference in human IgG2 disulfide isoforms revealed byhydrogen/deuterium exchange mass spectrometry”, Biochemistry, 54(10),1956-1962; FIG. 10 ). These isoforms were assessed by RP-HPLC (FIG. 10), based on methods developed by Dillon et al., “Optimization of areversed-phase high-performance liquid chromatography/mass spectrometrymethod for characterizing recombinant antibody heterogeneity andstability”, J Chromatography A, 1120(1), 112-120. The optimized methodused a shallower and higher organic mobile phase B content relative tothat in Dillon (id). Separate A and B forms enriched from INX901 wereprepared closely following the conditions reported in Dillon (id) butcombined with a buffer exchange back into DPBS and an endotoxin removalprocedure employed subsequent to the enrichment reactions (FIG. 11 ).

In the course of preparing these experiments it was observed thatreversion of the A-enriched form occurs more quickly than expected, andat lower residual redox reagent concentrations than expected.Utilization of a fast-spin, size-exclusion based desalting procedure wastherefore employed, which appeared to largely prevent this reversion. Asshown in panel (A) in FIG. 10 disulfide shuffling leads to isoforms Aand B, along with the transition for A/B (reproduced from Zhang, A. etal., 2015). (B) Isoforms are distinguishable by RP-HPLC (figure fromZhang, A. et al., 2015). (C) Observed RP-HPLC chromatogram for INX901.

The inventors optimized RP-HPLC Method for detecting IgG2 isoforms isdescribed below. In FIG. 11 : (Black line, top) the chromatogram shows adominant left-most peak defining the B-form. (Red line, bottom)Chromatogram shows a dominant right pea k defining the A-form.

Optimized RP-HPLC Methods for Isoform Detection

Mobile Phase A Preparation (0.1% v/v TFA in Water):

-   -   1. Measured 1.0 L Milli-Q water in a 1.0 L graduated cylinder    -   2. Added 1.0 mL of TFA to the 1 L of water using a 1 mL glass        Hamilton syringe    -   3. Transferred the solution to a 1 L bottle, mixed well.    -   4. Expiry is 2 weeks after preparation        Mobile Phase B Preparation (70% v/v IPA, 20% v/v ACN, 9.9% v/v        water, 0.1% v/v TFA):    -   1. Measured 700 mL IPA into a 1.0 L graduated cylinder    -   2. Measured 200 mL ACN into a 250 mL graduated cylinder and        transferred to the 1.0 L graduated cylinder containing the 700        mL IPA    -   3. Added Milli-Q water to the 1.0 L graduated cylinder        containing the 700 mL IPA and 200 mL ACN until the liquid        reached to 1.0 L mark    -   4. Added 1.0 mL of TFA to the 1 L of water using a 1 mL glass        Hamilton syringe    -   5. Transferred the solution to a 1 L bottle, mixed well.    -   6. Expiry is 2 weeks after preparation        RP-HPLC Chromatography Conditions    -   1. Column A (large bore): Zorbax 300SB-C8, 5μηη, 2.1×150 mm,        «OR»,    -   2. Column B (narrow bore): Zorbax 300SB-C8, 3.5μηη, 1×50 mm    -   3. Mobile Phase A: 0.1% v/v TFA in water    -   4. Mobile Phase B: 70% v/v IPA, 20% v/v ACN, 9.9% v/v water,        0.1% v/v TFA    -   5. Flow rate: 0.5 m L/min for Column A or 0.25 m L/min for        Column B    -   6. Column compartment: 75.0±1.0° C.    -   7. Detection: 214 nm    -   8. RP-HPLC mobile phase gradient (Table below)

Time (min) Mobile Phase B %  0 15  2 26 34 36 35 75 36 15 40 15INX901 Disulfide Isoform Enrichment MethodsB-Form Enrichment

-   -   1. Into endotoxin free non-pyrogenic tube, add:        -   2.1 mL of INX901 (5.66 mg/mL)        -   792.6 μL 1 M Tris pH 8.0        -   495.4 μL endo-free water        -   396.3 additional endo-free water        -   237.8 μL of 100 mM Cysteine        -   39.6 μL of 100 mM Cystamine    -   2. Finger vortex (lightly), then place capped at 2-8° C. for 24        hr    -   3. Soaked Pall microsep spin-concentrator in 0.3M NaOH 2 hr at        RT, then rinsed 3× with 10×DPBS, then 3× with endo-free water.        Air dried in BSC before use    -   4. Followed vendor's instructions for regenerating 0.5 mL        endotoxin removal column, using the 0.2N NaOH/95% ethanol (2 hrs        at RT) option for step 3; used 1×DPBS as final equilibration        buffer    -   5. Concentrated ˜4,020 μL of reaction (from Step 2) in a        separate PALL microsep (as prepared above).    -   6. Concentrated at 2,500×G for 35 min to less 0.4 mL (>10×) then        re-diluted with 4 mL I×DPBS, repeated 2 additional times    -   7. Concentrated at 2,500×G for 15 min to below 2 mL, then added        back I×DPBS to 2 mL    -   8. Added all 2 mL of buffer exchanged sample to the regenerated,        spun dried, bottom capped endotoxin removal column, capped the        top tightly, inverted, placed at room temp-inverted 3 more times        every ˜20 minutes, then spun out the sample into non-pyrogenic        tube (1 min at 500×G, as per Vendor's instructions), placed at        2-8° C.        A-Form Enrichment    -   1. Into endotoxin free non-pyrogenic tube, add:        -   1750 μL INX901 (6.2 mg/mL)        -   370 μL endo-free water        -   700 μL I M Tris pH8.0        -   435 μL 8M GdCl        -   210 μL 0.1 M Cysteine HCl (made fresh from 1 M stock)        -   35 μL 0.1 M Cystamine-2HCl (made fresh from 1 M stock)        -   (Final volume 3500 μL)    -   2. Finger vortex (lightly), then place capped at 2-8° C. for 24        hr    -   3. Prepared #7-2 mL Zeba spin columns (Thermo P/N 89890) as per        vendor's instructions, equilibrating into 1× Dulbecco's        Phosphate Buffered Saline (DPBS).    -   4. Loaded 500 μL of the above reaction mixture onto each of the        #7, and spun 2 minutes at 1000×G (also as per vendor's        instructions), collecting into clean pyrogen free tubes.    -   5. Placed in de-pyrogenated PALL microsep, spun total of 1 hour,        10 minutes, concentrated to approximately 1.7 mL at        approximately 5 mg/mL    -   6. Added all ˜1.7 mL above to one 0.5 mL endotoxin removal spin        column (Thermo P/N 88274) prepared as per Vendor's instructions        (including overnight in 0.2 M NaOH at room tempo), equilibrated        into 1×DPBS. Left at room temp approximately 1 hr, then placed        at 4° C. for approximately another 1 hr, in both cases inverting        the capped tube about every 15 minutes.    -   7. Recovered prep by spinning 500×G for 1 minute (also as per        vendor's instructions).    -   8. Recovered volume: approximately 1.3 mL at 4.61 mg/mL (all        concentrations based on the NanoDrop's built-in IgG extinction        coefficient of 0.73)

IgG2 A- and B-Locked Variants

Specific substitutions to the amino acid sequence of IgG2 are capable ofpreventing disulfide shuffling, and depending on the mutation willresult in a locked conformation that is either A-like or B-like(Martinez, et al., (2008). “Disulfide connectivity of humanimmunoglobulin G2 structural isoforms”, Biochemistry, 47(28), 7496-7508;Allen, et al., (2009), “Interchain disulfide bonding in human IgG2antibodies probed by site-directed mutagenesis”, Biochemistry, 48(17),3755-3766.

The inventors therefore designed INX901 and INX908 variants with eitherthe C233S (A-locked) or C127S (B-locked) mutation (Eu numbering) tomatch the IgG2 variants used by White et al., (2015), “Conformation ofthe human immunoglobulin G2 hinge imparts superagonistic properties toimmunostimulatory anticancer antibodies”, Cancer Cell, 27(1), 138-148.Constant heavy chain sequences are listed below.

IgG2 C233S (A-locked) (SEQ ID NO: 63)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK IgG2 C127S (B-locked)(SEQ ID NO: 64) ASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSilent Fc Variants

The inventors designed INX901 and INX908 variants with a silent Fcregion by introducing the following point mutations on an IgGI backbone:L234A/L235A/G237A/P238A/H268A/A330S/P331S (McCarthy et al., (2015) U.S.patent application Ser. No. 14/818,864. Washington, D.C.: U.S. In onetype of variant (INX901Si and INX908Si), the CHI/hinge region of theheavy constant region is native IgGI, which does not support thedisulfide shuffling of a native IgG2 (FIG. 12 , middle). In a secondtype of variant (INX901HSi and INX908HSi), the CHI/hinge region isnative IgG2, which does support disulfide shuffling (White, A. L. etal., 2015) (FIG. 12 , bottom). Constant heavy chain sequences for bothtypes of variants are listed below.

IgG I with silent Fc (INX901Si and INX908Si) (SEQ ID NO: 65)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK IgG2 CHI/hinge +IgGI silent Fc (INX901HSi and INX908HSi) (SEQ ID NO: 66)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The experiments in FIG. 12 compare the immune properties of INX901Fc-silent variants with respect to disulfide shuffling. (Top) INX901 onan IgG2 backbone exhibits an expected mixture of A, A/B, and B isoforms.(Middle) INX901Si on a silent IgGI backbone exists as a single isoform.(Bottom) INX901HSi possesses an IgGI silent Fc region with a CHI/hingefrom IgG2, which enables disulfide shuffling equivalent to native IgG2.These results indicate that FcR binding appears to affect the agonistproperties of the inventive antibodies.

Example 15: Function of INX901 and INX908 in Various Ig Backbones toDetermine Requirement of Hinge and Fc Regions

We conducted experiments to assess the functional requirements of theCHI/hinge and Fc regions of the heavy chain of the anti-human VISTAantibodies, INX901 and INX908. In their original state, both moleculesare on native human IgG2 backbones, and are therefore mixtures ofconformationally distinct isoforms resulting disulfide shuffling. Thehigh cell density mixed lymphocyte reaction (MLR) was chosen for thesestudies as previous data indicates that this assay provides a robustread out of functionality for both INX901 and INX908. The followingmodifications of INX901 and/or INX908 were made to investigate whetherspecific isoforms are responsible for function: biochemical skewing toeither the A or B isoform, genetic modifications to “lock” theconformation into the A or B form, and chimeric molecules where the Fcwas silenced and the CHI/hinge region came from either IgGI, in whichdisulfide shuffling does not occur, or IgG2, which allows for nativedisulfide shuffling.

The results of the assay indicate that INX901 and INX908 retain functionregardless of whether in the A form, B form, or the mixture of formsthat characterizes a native IgG2. Additionally, both INX901 and INX908require an active Fc region for functionality.

The MLR is a standard immunological assay that depends upon MHC classland II mismatching to drive an allogeneic T cell response. Peripheralblood mononuclear cells are isolated from two mismatched individuals,incubated together and as a result of these mismatches, proliferationand cytokine production occurs. High cell density conditions (HCD),meaning cultures with >I×I0⁷ cells/ml, have previously been reported toelucidate agonistic functions of antibodies in vitro. Our previous dataindicates that both INX901 and INX908 can suppress the expression ofTNFa under HCD conditions in the MLR.

The HCD MLR assay was used to assess the function of INX901 and INX908following either genetic or biochemical modifications with respect toIgG2 disulfide isoforms and/or Fc silencing of each antibody. Prior torunning the MLR, each antibody was confirmed to bind recombinant VISTAvia ELISA. INX901 was sent to Elion, LLC (Louisville, Colo.) where itwas modified by redox to either be predominantly A form (INX901 A skew)or B form (INX901 B skew). Skewing was confirmed by RP-HPLC as describedin the prior example. (FIG. 11 ). Each antibody, as well as the parentalINX901, was diluted in a dose response in the HCD MLR and cytokineproduction was measured by Luminex. Previous data has indicated thatTNFa and/or IL-2 are robust readouts for antibody function of theparental INX901 antibody. In two separate MLRs, both TNFa and IL-2 werereduced by INX901 parental, INX901 A skew and INX901 B skew compared tothe IgG2 control (FIG. 13 ).

To confirm the data from FIG. 13 , additional variants of INX901 weremade with mutations to generate locked variants in either the A form orthe B form. Additionally, chimeric versions of INX901 were made withfully silent Fc regions to test the function of the Fc domain. INX901 Siis a fully silent IgGI antibody. INX901 HSi has a fully silent IgGI Fc,but also possesses an IgG2 CHI/hinge region that enables disulfideshuffling that is indistinguishable from a native IgG2. Prior to runningthe MLR, each antibody was confirmed to bind recombinant VISTA viaELISA. Confirming the data from the biochemical skewing, both the A lockand B locked versions of INX901 were able to reduce the production ofboth IL-2 and TNFa (FIG. 14 ). In contrast, both the Si and HSi versionsof INX901 were unable to reduce production of IL-2 and TNFa (FIG. 14 ).

To confirm the data from FIG. 14 , identical mutations were made to theINX908 antibody to generate locked variants in either the A form or theB form. Additionally, chimeric versions of INX908 were made with fullysilent Fc regions to test the function of the Fc domain. INX908 Si is afully silent IgGI antibody. INX908 HSi has a fully silent IgGI Fc butcontains the IgG2 CHI/hinge region. Prior to running the MLR, eachantibody was confirmed to bind recombinant VISTA via ELISA. Confirmingthe data with the INX901 variants, both the A lock and B locked versionsof INX908 were able to reduce the production of both IL-2 and TNFa (FIG.15 ). In contrast, both the Si and HSi versions of INX908 were unable toreduce production of IL-2 and TNFa (FIG. 15 ).

Example 15: Discontinuous Epitope Mapping of Agonist Antibodies UsingPEPPSCAN Methods

Pepscan uses peptide arrays to determine both linear and discontinuousepitopes. This methodology is an accepted method used by manyresearchers and companies to ascertain antibody epitopes. FIG. 16schematically describes the Pepscan® technology used to identify linearand discontinuous epitopes bound by various agonist anti-human VISTAantibodies according to the invention.

The Principles of CLIPS Technology

CLIPS technology structurally fixes peptides into definedthree-dimensional structures. This results in functional mimics of eventhe most complex binding sites. CLIPS technology is now routinely usedto shape peptide libraries into single, double or triple loopedstructures as well as sheet- and helix-like folds. The CLIPS reactiontakes place between bromo groups of the CLIPS scaffold and thiolsidechains of cysteines. The reaction is fast and specific under mildconditions. Using this elegant chemistry, native protein sequences aretransformed into CLIPS constructs with a range of structures.

Combinatorial CLIPS Library Screening in Detail

CLIPS library screening starts with the conversion of the target proteininto a library of up to 10,000 overlapping peptide constructs, using acombinatorial matrix design. On a solid carrier, a matrix of linearpeptides is synthesized, which are subsequently shaped into spatiallydefined CLIPS constructs. Constructs representing both parts of thediscontinuous epitope in the correct conformation bind the antibody withhigh affinity, which is detected and quantified. Constructs presentingthe incomplete epitope bind the antibody with lower affinity, whereasconstructs not containing the epitope do not bind at all. Affinityinformation is used in iterative screens to define the sequence andconformation of epitopes in detail. The results of this epitope analysisare summarized below.

Antibodies INX901, INX902, INX904, INX906, INX907, INX908

When tested under moderate stringency conditions antibodies INX901,INX902, INX904, INX906, INX907, INX908 strongly bound linear andconformational epitope mimics. Bound peptides contain core sequences₄₈NVTLTCRLLGPV₆₀ (SEQ ID NO:67), ₇₉EVQTCSERRPIR₉₀ (SEQ ID NO:68),123SDHHGNFS130 (SEQ ID NO:69) and i₅₃HHHSEHi58 (SEQ ID NO:70), wherepeptide stretch 79EVQTCSERRPIR90 (SEQ ID NO:68) is the dominant part ofthe epitope.

Additional analysis of data recorded with linear epitope mimics allowedus to identify residues that are important for binding for INX 904,INX906, INX907 and INX908, as double Ala mutants on certain positionsnotably decreased signal intensities. In particular, replacement ofresidues CR within ₄₈NVTLTCRLLGPV₆₀ (SEQ ID NO:71) affects binding ofINX906, INX907 and INX908. Also the replacement of residues TC within79EVQTCSERRPIR90 (SEQ ID NO:68) notably affects binding of INX904 andINX907.

Antibody INX800

When tested under moderate stringency conditions antibody INX800 did notdetectably bind linear and simple constrained epitope mimics, but showeddetectable binding with discontinuous epitope mimics. Analysis of dataobtained with discontinuous epitope mimics suggest that antibody INX800recognizes a discontinuous epitope with core sequences ₅₃TCRLLGPVDKG₆₃(SEQ ID NO:72), 101HGGHQAAi₀₇ (SEQ ID NO:73), i2iSASDHHGNFSi3o (SEQ IDNO:74) and _(i53)HHHSEHRVHGAMi₆₄ (SEQ ID NO:75), where sequence₁₅₃HHHSEHRVHGAM₁₆₄ (SEQ ID NO:76) represents the dominant recognitionsite.

Antibodies INX803 and INX804

When tested under high stringency conditions antibodies INX803 andINX804 did not bind any peptide present on the array. When tested undermoderate stringency conditions both antibodies bound discontinuousepitope mimics. Cumulative analysis of binding profiles suggests thatboth antibodies similarly recognize peptide stretches ₅₂LTCRLLGPV₆₀ (SEQID NO:77), ₇₉EVQTCSERRPIR_(g0) (SEQ ID NO:78), ₉₈HLHHGGHQAAio7 (SEQ IDNO:79), _(i23)SDHHGNFSi3o {SEQ ID NO:80), i₅₃HHHSEHRVHGAMi₆₄ (SEQ IDNO:81), where region ₅₂LTCRLLGPV60 (SEQ ID NO:77) is the dominantrecognition site.

Antibody INX900

When tested under high stringency conditions antibody INX900 very weaklybound linear epitope mimics with core sequence ygEVQTCSERRPI_(Rgo) iSEQID NO:68). Notably higher binding was observed with discontinuousepitope mimics, which in addition to sequence ygEVQTCSERRPI_(Rgo) iSEQID NO:68) contain core sequences ₅₆LLGPVDKGHDVTFYKyo (SEQ ID NO:82),ii₃LAQRHGLESASDHHGi₂y (SEQ ID NO:83), _(i53)HHHSEHRVHGAMi₆₄ (SEQ IDNO:84).

Antibody INX903

When tested under high stringency conditions antibody INX903 did notbind linear epitope mimics, but weakly bound conformational epitopemimics. Analysis of recorded intensity profiles suggests that theantibody recognizes a discontinuous epitope composed of core sequencesy₉EVQTCSERR₈₇ (SEQ ID NO:85), ₉₃TFQDLHLHHGGHQAA₁₀₇ (SEQ ID NO:86),i₄₆CLVVEIRHHHSEHi₅₈ (SEQ ID NO:87), where sequence ₇₉EVQTCSERR₈₇ (SEQ IDNO:85) is the core of the epitope.

Antibody INX905

When tested under high stringency conditions antibody INX905 boundlinear peptides with core sequence ₇₉EVQTCSERRP₈₈ (SEQ ID NO:88). Dataacquired with double Ala mutants indicate that motif RR within₇₉EVQTCSERRP₈₈ (SEQ ID NO:88) is critical for the recognition. Intensityprofiles recorded with discontinuous epitope mimics suggest thataddition of peptide sequences ₅₃TCRLLGPVDKG₆₃ (SEQ ID NO:89),i₂₃SDHHGi₂₇ (SEQ ID NO:90) and i₅₃HHHSEHRVHGAMi₆₄ (SEQ ID NO:91)augments binding of the antibody. FIG. 17 shows that most agonistanti-human VISTA antibodies bind to the same core sequence. FIG. 18 alsosummarizes the epitope results. FIG. 19 shows the epitopes bound byexemplary agonist anti-human VISTA antibodies according to the inventionand identifies important residues involved in binding.

REFERENCES CITED IN THIS APPLICATION

The following references and other references cited in this applicationare incorporated by reference in their entireties.

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SEQUENCE LISTINGSEQ ID NO: 1: Homo sapiens VISTA (Alternate names: B7-H5; B7H5; DDIal pha;GI24; PP2135; SISP1) AMINO ACID SEQUENCE 1mgvpta leag swrwgsl lfa lflaaslgpv aafkvatpys lyvcpegq nv tltcrl lgpv 61d kghdvtfyk twyrssrgev qtcserrpir nltfqd lhlh hgghqaa nts hd laqrhgle121sasd hhgnfs itm rnltl ld sglycclwe irhh hseh rv hgamelqvqt gkda psncvv181ypsssqdsen itaaa latga civgilcl pl ill lvykq rq aasnrraqel vrmdsniqgi241 enpgfeaspp aqgipea kvr hplsyvaqrq psesgrhl ls epstplsppg pgdvffpsld301 pvpdspnfev i SEQ ID NO: 2: Mus muscu lus VISTA AMINO ACID SEQUENCE 1mgvpavpeas sprwgtl lla iflaasrglv aafkvttpys lyvcpegq na tltcrilgpv 61skghdvtiyk twylssrgev qmckehrpir nftlq hlq h h gshl ka nash dqpq khglel121asd h hgnfsi t lrnvtprds glycclviel knh hpeqrfy gsmelqvqag kgsgstcmas181neqdsdsita aa latgaciv gilcl plil l lvykqrqvas hrraqelvrm dsntqgien p241gfettppfqg mpea ktrppl syvaq rq pse sgryl lsdps tplsppgpgd vffpsld pvp301 dspnseai SEQ ID NO: 3: Mus muscu lus VISTA AMINO ACID SEQUENCE 1mgvpavpeas sprwgtl lla iflaasrglv aafkvttpys lyvcpegq na tltcrilgpv 61skghdvtiyk  twylssrgev q mckehrpir nftlq hlq h h gshl ka nash dqpq khglel121asd h hgnfsi t lrnvtprds glycclviel knh hpeqrfy gsmelqvqag kgsgstcmas181 neqdsdsita aa latgaciv gilcl plil l lvykqrqvas hrraqelvrm dssntqgien241pgfettppfq g m pea ktrpp lsyvaq rqps esgryllsd p stplsppgpg dvffpsld pv301 pdspnseaiSEQ ID NO: 4: Homo sapiens VISTA (Alternate names: B7-H5; B7H5; DDlalpha; 0l24; PP2135; SISP1) NUCLEIC ACID SEQUENCE 1gggggcgggt gcctggagca cggcgctggg gccgcccgca gcgctcactc gctcgcactc 61agtcgcggga ggcttccccg cgccggccgc gtcccgcccg ctccccggca ccagaagttc 121ctctgcgcgt ccgacggcga catgggcgtc cccacggccc tggaggccgg cagctggcgc 181tggggatccc tgctcttcgc tctcttcctg gctgcgtccc taggtccggt ggcagccttc 241aaggtcgcca cgccgtattc cctgtatgtc tgtcccgagg ggcagaacgt caccctcacc 301tgcaggctct tgggccctgt ggacaaaggg cacgatgtga ccttctacaa gacgtggtac 361cgcagctcga ggggcgaggt gcagacctgc tcagagcgcc ggcccatccg caacctcacg 421ttccaggacc ttcacctgca ccatggaggc caccaggctg ccaacaccag ccacgacctg 481gctcagcgcc acgggctgga gtcggcctcc gaccaccatg gcaacttctc catcaccatg 541cgcaacctga ccctgctgga tagcggcctc tactgctgcc tggtggtgga gatcaggcac 601caccactcgg agcacagggt ccatggtgcc atggagctgc aggtgcagac aggcaaagat 661gcaccatcca actgtgtggt gtacccatcc tcctcccagg atagtgaaaa catcacggct 721gcagccctgg ctacgggtgc ctgcatcgta ggaatcctct gcctccccct catcctgctc 781ctggtctaca agcaaaggca ggcagcctcc aaccgccgtg cccaggagct ggtgcggatg 841gacagcaaca ttcaagggat tgaaaacccc ggctttgaag cctcaccacc tgcccagggg 901atacccgagg ccaaagtcag gcaccccctg tcctatgtgg cccagcggca gccttctgag 961tctgggcggc atctgctttc ggagcccagc acccccctgt ctcctccagg ccccggagac 1021gtcttcttcc catccctgga ccctgtccct gactctccaa actttgaggt catctagccc 1081agctggggga cagtgggctg ttgtggctgg gtctggggca ggtgcatttg agccagggct 1141ggctctgtga gtggcctcct tggcctcggc cctggttccc tccctcctgc tctgggctca 1201gatactgtga catcccagaa gcccagcccc tcaacccctc tggatgctac atggggatgc 1261tggacggctc agcccctgtt ccaaggattt tggggtgctg agattctccc ctagagacct 1321gaaattcacc agctacagat gccaaatgac ttacatctta agaagtctca gaacgtccag 1381cccttcagca gctctcgttc tgagacatga gccttgggat gtggcagcat cagtgggaca 1441agatggacac tgggccaccc tcccaggcac cagacacagg gcacggtgga gagacttctc 1501ccccgtggcc gccttggctc ccccgttttg cccgaggctg ctcttctgtc agacttcctc 1561tttgtaccac agtggctctg gggccaggcc tgcctgccca ctggccatcg ccaccttccc 1621cagctgcctc ctaccagcag tttctctgaa gatctgtcaa caggttaagt caatctgggg 1681cttccactgc ctgcattcca gtccccagag cttggtggtc ccgaaacggg aagtacatat 1741tggggcatgg tggcctccgt gagcaaatgg tgtcttgggc aatctgaggc caggacagat 1801gttgccccac ccactggaga tggtgctgag ggaggtgggt ggggccttct gggaaggtga 1861gtggagaggg gcacctgccc cccgccctcc ccatccccta ctcccactgc tcagcgcggg 1921ccattgcaag ggtgccacac aatgtcttgt ccaccctggg acacttctga gtatgaagcg 1981ggatgctatt aaaaactaca tggggaaaca ggtgcaaacc ctggagatgg attgtaagag 2041ccagtttaaa tctgcactct gctgctcctc ccccaccccc accttccact ccatacaatc 2101tgggcctggt ggagtcttcg cttcagagcc attcggccag gtgcgggtga tgttcccatc 2161tcctgcttgt gggcatgccc tggctttgtt tttatacaca taggcaaggt gagtcctctg 2221tggaattgtg attgaaggat tttaaagcag gggaggagag tagggggcat ctctgtacac 2281tctgggggta aaacagggaa ggcagtgcct gagcatgggg acaggtgagg tggggctggg 2341cagaccccct gtagcgttta gcaggatggg ggccccaggt actgtggaga gcatagtcca 2401gcctgggcat ttgtctccta gcagcctaca ctggctctgc tgagctgggc ctgggtgctg 2461aaagccagga tttggggcta ggcgggaaga tgttcgccca attgcttggg gggttggggg 2521gatggaaaag gggagcacct ctaggctgcc tggcagcagt gagccctggg cctgtggcta 2581cagccaggga accccacctg gacacatggc cctgcttcta agccccccag ttaggcccaa 2641aggaatggtc cactgagggc ctcctgctct gcctgggctg ggccaggggc tttgaggaga 2701gggtaaacat aggcccggag atggggctga cacctcgagt ggccagaata tgcccaaacc 2761ccggcttctc ccttgtccct aggcagaggg gggtcccttc ttttgttccc tctggtcacc 2821acaatgcttg atgccagctg ccataggaag agggtgctgg ctggccatgg tggcacacac 2881ctgtcctccc agcactttgc agggctgagg tggaaggacc gcttaagccc aggtgttcaa 2941ggctgctgtg agctgtgttc gagccactac actccagcct ggggacggag caaaactttg 3001cctcaaaaca aattttaaaa agaaagaaag aaggaaagag ggtatgtttt tcacaattca 3061tgggggcctg catggcagga gtggggacag gacacctgct gttcctggag tcgaaggaca 3121agcccacagc ccagattccg gttctcccaa ctcaggaaga gcatgccctg ccctctgggg 3181aggctggcct ggccccagcc ctcagctgct gaccttgagg cagagacaac ttctaagaat 3241ttggctgcca gaccccaggc ctggctgctg ctgtgtggag agggaggcgg cccgcagcag 3301aacagccacc gcacttcctc ctcagcttcc tctggtgcgg ccctgccctc tcttctctgg 3361acccttttac aactgaacgc atctgggctt cgtggtttcc tgttttcagc gaaatttact 3421ctgagctccc agttccatct tcatccatgg ccacaggccc tgcctacaac gcactaggga 3481cgtccctccc tgctgctgct ggggaggggc aggctgctgg agccgccctc tgagttgccc 3541gggatggtag tgcctctgat gccagccctg gtggctgtgg gctggggtgc atgggagagc 3601tgggtgcgag aacatggcgc ctccaggggg cgggaggagc actaggggct ggggcaggag 3661gctcctggag cgctggattc gtggcacagt ctgaggccct gagagggaaa tccatgcttt 3721taagaactaa ttcattgtta ggagatcaat caggaattag gggccatctt acctatctcc 3781tgacattcac agtttaatag agacttcctg cctttattcc ctcccaggga gaggctgaag 3841gaatggaatt gaaagcacca tttggagggt tttgctgaca cagcggggac tgctcagcac 3901tccctaaaaa cacaccatgg aggccactgg tgactgctgg tgggcaggct ggccctgcct 3961gggggagtcc gtggcgatgg gcgctggggt ggaggtgcag gagccccagg acctgctttt 4021caaaagactt ctgcctgacc agagctccca ctacatgcag tggcccaggg cagaggggct 4081gatacatggc ctttttcagg gggtgctcct cgcggggtgg acttgggagt gtgcagtggg 4141acagggggct gcaggggtcc tgccaccacc gagcaccaac ttggcccctg gggtcctgcc 4201tcatgaatga ggccttcccc agggctggcc tgactgtgct gggggctggg ttaacgtttt 4261ctcagggaac cacaatgcac gaaagaggaa ctggggttgc taaccaggat gctgggaaca 4321aaggcctctt gaagcccagc cacagcccag ctgagcatga ggcccagccc atagacggca 4381caggccacct ggcccattcc ctgggcattc cctgctttgc attgctgctt ctcttcaccc 4441catggaggct atgtcaccct aactatcctg gaatgtgttg agagggattc tgaatgatca 4501atatagcttg gtgagacagt gccgagatag atagccatgt ctgccttggg cacgggagag 4561ggaagtggca gcatgcatgc tgtttcttgg ccttttctgt tagaatactt ggtgctttcc 4621aacacacttt cacatgtgtt gtaacttgtt tgatccaccc ccttccctga aaatcctggg 4681aggttttatt gctgccattt aacacagagg gcaatagagg ttctgaaagg tctgtgtctt 4741gtcaaaacaa gtaaacggtg gaactacgac taaa //SEQ ID NO: 5: Homo sapiens VISTA (Alternate names: B7-H5; B7H5; DDlalpha; Gl24; PP2135; SlSP1) CODING NUCLEIC ACID SEQUENCE 1ctcgccgcgc tgagccgcct cgggacggag ccatgcggcg ctgggcctgg gccgcggtcg 61tggtccccct cgggccgcag ctcgtgctcc tcgggggcgt cggggcccgg cgggaggcac 121agaggacgca gcagcctggc cagcgcgcag atccccccaa cgccaccgcc agcgcgtcct 181cccgcgaggg gctgcccgag gcccccaagc catcccaggc ctcaggacct gagttctccg 241acgcccacat gacatggctg aactttgtcc ggcggccgga cgacggcgcc ttaaggaagc 301ggtgcggaag cagggacaag aagccgcggg atctcttcgg tcccccagga cctccaggtg 361cagaagtgac cgcggagact ctgcttcacg agtttcagga gctgctgaaa gaggccacgg 421agcgccggtt ctcagggctt ctggacccgc tgctgcccca gggggcgggc ctgcggctgg 481tgggcgaggc ctttcactgc cggctgcagg gtccccgccg ggtggacaag cggacgctgg 541tggagctgca tggtttccag gctcctgctg cccaaggtgc cttcctgcga ggctccggtc 601tgagcctggc ctcgggtcgg ttcacggccc ccgtgtccgg catcttccag ttctctgcca 661gtctgcacgt ggaccacagt gagctgcagg gcaaggcccg gctgcgggcc cgggacgtgg 721tgtgtgttct catctgtatt gagtccctgt gccagcgcca cacgtgcctg gaggccgtct 781caggcctgga gagcaacagc agggtcttca cgctacaggt gcaggggctg ctgcagctgc 841aggctggaca gtacgcttct gtgtttgtgg acaatggctc cggggccgtc ctcaccatcc 901aggcgggctc cagcttctcc gggctgctcc tgggcacgtg agggcgccca ggggggctgg 961cgaggagctg ccgccggatc ccggggaccc tcctactgat gcccgtggtc accacaataa 1021agagccctcc accctcaaaa aaaaaaaaaa aaaaa //SEQ ID NO: 6: Mus muscu lus VISTA CODING NUCLEIC ACID SEQUENCE 1ctcgccgcgc tgagccgcct cgggacggag ccatgcggcg ctgggcctgg gccgcggtcg 61tggtccccct cgggccgcag ctcgtgctcc tcgggggcgt cggggcccgg cgggaggcac 121agaggacgca gcagcctggc cagcgcgcag atccccccaa cgccaccgcc agcgcgtcct 181cccgcgaggg gctgcccgag gcccccaagc catcccaggc ctcaggacct gagttctccg 241acgcccacat gacatggctg aactttgtcc ggcggccgga cgacggcgcc ttaaggaagc 301ggtgcggaag cagggacaag aagccgcggg atctcttcgg tcccccagga cctccaggtg 361cagaagtgac cgcggagact ctgcttcacg agtttcagga gctgctgaaa gaggccacgg 421agcgccggtt ctcagggctt ctggacccgc tgctgcccca gggggcgggc ctgcggctgg 481tgggcgaggc ctttcactgc cggctgcagg gtccccgccg ggtggacaag cggacgctgg 541tggagctgca tggtttccag gctcctgctg cccaaggtgc cttcctgcga ggctccggtc 601tgagcctggc ctcgggtcgg ttcacggccc ccgtgtccgg catcttccag ttctctgcca 661gtctgcacgt ggaccacagt gagctgcagg gcaaggcccg gctgcgggcc cgggacgtgg 721tgtgtgttct catctgtatt gagtccctgt gccagcgcca cacgtgcctg gaggccgtct 781caggcctgga gagcaacagc agggtcttca cgctacaggt gcaggggctg ctgcagctgc 841aggctggaca gtacgcttct gtgtttgtgg acaatggctc cggggccgtc ctcaccatcc 901aggcgggctc cagcttctcc gggctgctcc tgggcacgtg agggcgccca ggggggctgg 961cgaggagctg ccgccggatc ccggggaccc tcctactgat gcccgtggtc accacaataa 1021agagccctcc accctcaaaa aaaaaaaaaa aaaaa //

The invention claimed is:
 1. An anti-human antibody or antibody fragmentthereof comprising an antigen binding region that specifically binds tohuman V-domain Ig Suppressor of T cell Activation (human VISTA), whereinthe variable heavy (VH) polypeptide of the antibody or antibody fragmentcomprises the VH complementarity determining region-1 (CDR1), CDR2 andCDR3 polypeptides of SEQ ID NO: 400, 401 and 402, respectively, and thevariable light (VL) polypeptide of the antibody or antibody fragmentcomprises the VL CDR1, CDR2 and CDR3 polypeptides of SEQ ID NO: 403, 404and 405, respectively.
 2. The anti-human antibody or antibody fragmentof claim 1, which comprises a VH polypeptide possessing at least 90%sequence identity to SEQ ID NO:
 406. 3. The anti-human antibody orantibody fragment of claim 1, which comprises a VH polypeptidepossessing at least 95% sequence identity to SEQ ID NO:
 406. 4. Theanti-human antibody or antibody fragment of claim 1, which comprises aVH polypeptide possessing sequence identity to SEQ ID NO:
 406. 5. Theanti-human antibody or antibody fragment of claim 1, which comprises aVL polypeptide possessing at least 90% sequence identity to SEQ ID NO:408.
 6. The anti-human antibody or antibody fragment of claim 1, whichcomprises a VL polypeptide possessing at least 95% sequence identity toSEQ ID NO:
 408. 7. The anti-human antibody or antibody fragment of claim1, which comprises a VL polypeptide possessing sequence identity to SEQID NO:
 408. 8. The anti-human antibody or antibody fragment of claim 1,which comprises a VH polypeptide possessing at least 90% sequenceidentity to SEQ ID NO: 406, and a VL polypeptide possessing at least 90%sequence identity to SEQ ID NO:
 408. 9. The anti-human antibody orantibody fragment of claim 1, which comprises a VH polypeptidepossessing at least 95% sequence identity to SEQ ID NO: 406, and a VLpolypeptide possessing at least 95% sequence identity to SEQ ID NO: 408.10. The anti-human antibody or antibody fragment of claim 1, whichcomprises a VH polypeptide possessing sequence identity to SEQ ID NO:406, and a VL polypeptide possessing identity to SEQ ID NO:
 408. 11. Theanti-human antibody or antibody fragment of claim 1, which comprises ahuman constant region.
 12. The anti-human antibody or antibody fragmentof claim 1, which comprises a human IgG1, IgG2, IgG3 or IgG4 constantregion.
 13. The anti-human antibody or antibody fragment of claim 1,which comprises a human IgG1, IgG2, IgG3 or IgG4 constant region whichcomprises a mutation which impairs FcR binding.
 14. The anti-humanantibody or antibody fragment of claim 1, which comprises a human IgG1,IgG2, IgG3 or IgG4 constant region which comprises a mutation whichimpairs complement binding.
 15. The anti-human antibody or antibodyfragment of claim 1, which comprises a human IgG1, IgG2, IgG3 or IgG4constant region which comprises a mutation which impairs FcR binding anda mutation which impairs complement binding.
 16. The anti-human antibodyor antibody fragment of claim 1, which comprises a human IgG2 constantregion which is not modified.
 17. A composition comprising an anti-humanantibody or antibody fragment according to claim 1, and apharmaceutically acceptable carrier.
 18. A composition comprising ananti-human antibody or antibody fragment according to claim 12, and apharmaceutically acceptable carrier.
 19. A composition comprising ananti-human antibody or antibody fragment according to claim 15, and apharmaceutically acceptable carrier.
 20. A composition comprising ananti-human antibody or antibody fragment according to claim 16, and apharmaceutically acceptable carrier.